Assessing anaerobic gut fungal (Neocalliamstigomycota) diversity using PacBio D1/D2 LSU rRNA amplicon sequencing and multi-year isolation

The anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tracts of herbivores where they play a central role in the breakdown of ingested plant material. Accurate assessment of AGF diversity has been hampered by inherent deficiencies of the internal transcribed spacer1 (ITS1) region as a phylogenetic marker. Here, we report on the development and implementation of the D1/D2 region of the large ribosomal subunit (D1/D2 LSU) as a novel marker for assessing AGF diversity in culture-independent surveys. Sequencing a 1.4-1.5 Kbp amplicon encompassing the ITS1-5.8S rRNA-ITS2-D1/D2 LSU region in the ribosomal RNA locus from fungal strains and environmental samples generated a reference D1/D2 LSU database for all cultured AGF genera, as well as the majority of candidate genera encountered in prior ITS1-based diversity surveys. Subsequently, a D1/D2 LSU-based diversity survey using long read PacBio SMRT sequencing technology was conducted on fecal samples from 21 wild and domesticated herbivores. Twenty-eight genera and candidate genera were identified in the 17.7 K sequences obtained, including multiple novel lineages that were predominantly, but not exclusively, identified in wild herbivores. Association between certain AGF genera and animal lifestyles, or animal host family was observed. Finally, to address the current paucity of AGF isolates, concurrent isolation efforts utilizing multiple approaches to maximize recovery yielded 216 isolates belonging to twelve different genera, several of which have no prior cultured-representatives. Our results establish the utility of D1/D2 LSU and PacBio sequencing for AGF diversity surveys, and the culturability of a wide range of AGF taxa, and demonstrate that wild herbivores represent a yet-untapped reservoir of AGF diversity.

Previously undetected super-spreading of Mycobacterium tuberculosis revealed by deep sequencing

Tuberculosis disproportionately affects the Canadian Inuit. To address this, it is imperative we understand transmission dynamics in this population. We investigate whether ‘deep’ sequencing can provide additional resolution compared to standard sequencing, using a well-characterized outbreak from the Arctic (2011–2012, 50 cases). Samples were sequenced to ~500–1000x and reads were aligned to a novel local reference genome generated with PacBio SMRT sequencing. Consensus and heterogeneous variants were identified and compared across genomes. In contrast with previous genomic analyses using ~50x depth, deep sequencing allowed us to identify a novel super-spreader who likely transmitted to up to 17 other cases during the outbreak (35% of the remaining cases that year). It is increasingly evident that within-host diversity should be incorporated into transmission analyses; deep sequencing may facilitate more accurate detection of super-spreaders and transmission clusters. This has implications not only for TB, but all genomic studies of transmission - regardless of pathogen.

Previously undetected superspreading of Mycobacterium tuberculosis revealed by deep sequencing

Tuberculosis disproportionately affects the Canadian Inuit. To address this, it is imperative we understand transmission dynamics in this population. We investigate whether ‘deep’ sequencing can provide additional resolution compared to standard sequencing, using a well-characterized outbreak from the Arctic (2011-2012, 50 cases). Samples were sequenced to ~500-1000x and reads were aligned to a novel local reference genome generated with PacBio SMRT sequencing. Consensus and heterogeneous variants were identified and compared across genomes. In contrast with previous genomic analyses using ~50x depth, deep sequencing allowed us to identify a novel super-spreader who likely transmitted to up to 17 other cases during the outbreak (35% of all cases that year). It is increasingly evident that within-host diversity should be incorporated into transmission analyses; deep sequencing can facilitate accurately detection of super-spreaders and corresponding transmission clusters. This has implications not only for TB, but all genomic studies of transmission - regardless of pathogen.

High-Resolution Profiling of Gut Bacterial Communities in an Invasive Beetle using PacBio SMRT Sequencing System

Dendroctonus valens, an invasive bark beetle, has caused severe damage to Chinese forests. Previous studies have highlighted the importance of the gut microbiota and its fundamental role in host fitness. Culture-dependent and culture-independent methods have been applied in analyzing beetles’ gut microbiota. The former method cannot present a whole picture of the community, and the latter mostly generates short read lengths that cannot be assigned to species. Here, the PacBio sequencing system was utilized to capture full-length 16S rRNA sequences in D. valens gut throughout its ontogeny. A total of eight phyla, 55 families, 102 genera, and 253 species were identified. Bacterial communities in colonized beetles have the greatest richness but the lowest evenness in all life stages, which is different from those in young larvae. Pseudomonas sp., Serratia liquefaciens possess high abundance throughout its ontogeny and may serve as members of the core bacteriome. A phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis predicted that gut microbiota in larvae are rich in genes involved in carbohydrate, energy metabolism. Gut microbiota in both larvae and colonized beetles are rich in xenobiotics and terpenoids biodegradation, which are decreased in dispersal beetles. Considering that the results are based mainly on the analysis of 16S rRNA sequencing and PICRUSt prediction, further confirmation is needed to improve the knowledge of the gut microbiota in D. valens and help to resolve taxonomic uncertainty at the species level.

Genomic analysis of ST88 community-acquired methicillin resistantStaphylococcus aureusin Ghana

BackgroundThe emergence and evolution of community-acquired methicillin resistantStaphylococcus aureus(CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as an “African” CA-MRSA clone. In this study, we employed whole genome sequencing to provide more information on the genetic background of ST88 CA-MRSA isolates from Ghana and to describe in detail ST88 CA-MRSA isolates in comparison with other MRSA lineages worldwide.MethodsWe first established a complete ST88 reference genome (AUS0325) using PacBio SMRT sequencing. We then used comparative genomics to assess relatedness among 17 ST88 CA-MRSA isolates recovered from patients attending Buruli ulcer treatment centres in Ghana, three non-African ST88s and 15 other MRSA lineages.ResultsWe show that Ghanaian ST88 forms a discrete MRSA lineage (harbouring SCCmec-IV [2B]). Gene content analysis identified five distinct genomic regions enriched among ST88 isolates compared with the otherS. aureuslineages. The Ghanaian ST88 isolates had only 658 core genome SNPs and there was no correlation between phylogeny and geography, suggesting the recent spread of this clone. The lineage was also resistant to multiple classes of antibiotics includingβ-lactams, tetracycline and chloramphenicol.DiscussionThis study reveals thatS. aureusST88-IV is a recently emerging and rapidly spreading CA-MRSA clone in Ghana. The study highlights the capacity of small snapshot genomic studies to provide actionable public health information in resource limited settings. To our knowledge this is the first genomic assessment of the ST88 CA-MRSA clone.

Complete Genome Sequence of Potential Probiotic Lactobacillus sp. HFC8, Isolated from Human Gut Using PacBio SMRT Sequencing

We report a 3.07-Mb complete genome sequence of a lactic acid bacterium, Lactobacillus sp. HFC8. The gene-coding clusters are predicated for probiotic characteristics, like bacteriocin production, cell adhesion, bile salt hydrolysis, lactose metabolism, autoaggregation, and tolerance to oxidative stress.