Application of Pathogen Discovery/Metagenomic Sequencing in CNS HIV

Our culture-independent nanopore shotgun metagenomic sequencing protocol on biopsies has the potential for same-day diagnostics of orthopaedic implant-associated infections (OIAI). As OIAI are frequently caused by Staphylococcus aureus, we included S. aureus genotyping and virulence gene detection to exploit the protocol to its fullest. The aim was to evaluate S. aureus genotyping, virulence and antimicrobial resistance genes detection using the shotgun metagenomic sequencing protocol. This proof of concept study included six patients with S. aureus-associated OIAI at Akershus University Hospital, Norway. Five tissue biopsies from each patient were divided in two: (1) conventional microbiological diagnostics and genotyping, and whole genome sequencing (WGS) of S. aureus isolates; (2) shotgun metagenomic sequencing of DNA from the biopsies. Consensus sequences were analysed using spaTyper, MLST, VirulenceFinder, and ResFinder from the Center for Genomic Epidemiology (CGE). MLST was also compared using krocus. All spa-types, one CGE and four krocus MLST results matched Sanger sequencing results. Virulence gene detection matched between WGS and shotgun metagenomic sequencing. ResFinder results corresponded to resistance phenotype. S. aureus spa-typing, and identification of virulence and antimicrobial resistance genes are possible using our shotgun metagenomics protocol. MLST requires further optimization. The protocol has potential application to other species and infection types.

Download Full-text

Metagenomic Analysis of the Fecal Archaeome in Suckling Piglets Following Perinatal Tulathromycin Metaphylaxis

Animals ◽

10.3390/ani11061825 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1825

Author(s):

Mohamed Zeineldin ◽

Ameer Megahed ◽

Benjamin Blair ◽

Brian Aldridge ◽

James Lowe

Keyword(s):

Sequencing Analysis ◽

Metagenomic Sequencing ◽

Post Intervention ◽

Gastrointestinal Microbiome ◽

Health And Wellbeing ◽

Suckling Piglets ◽

And Function ◽

Changes Over Time ◽

And Control ◽

The Impact

The gastrointestinal microbiome plays an important role in swine health and wellbeing, but the gut archaeome structure and function in swine remain largely unexplored. To date, no metagenomics-based analysis has been done to assess the impact of an early life antimicrobials intervention on the gut archaeome. The aim of this study was to investigate the effects of perinatal tulathromycin (TUL) administration on the fecal archaeome composition and diversity in suckling piglets using metagenomic sequencing analysis. Sixteen litters were administered one of two treatments (TUL; 2.5 mg/kg IM and control (CONT); saline 1cc IM) soon after birth. Deep fecal swabs were collected from all piglets on days 0 (prior to treatment), 5, and 20 post intervention. Each piglet’s fecal archaeome was composed of rich and diverse communities that showed significant changes over time during the suckling period. At the phylum level, 98.24% of the fecal archaeome across all samples belonged to Euryarchaeota. At the genus level, the predominant archaeal genera across all samples were Methanobrevibacter (43.31%), Methanosarcina (10.84%), Methanococcus (6.51%), and Methanocorpusculum (6.01%). The composition and diversity of the fecal archaeome between the TUL and CONT groups at the same time points were statistically insignificant. Our findings indicate that perinatal TUL metaphylaxis seems to have a minimal effect on the gut archaeome composition and diversity in sucking piglets. This study improves our current understanding of the fecal archaeome structure in sucking piglets and provides a rationale for future studies to decipher its role in and impact on host robustness during this critical phase of production.

Download Full-text

Identification of the Potential Role of the Rumen Microbiome in Milk Protein and Fat Synthesis in Dairy Cows Using Metagenomic Sequencing

Animals ◽

10.3390/ani11051247 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1247

Author(s):

Xin Wu ◽

Shuai Huang ◽

Jinfeng Huang ◽

Peng Peng ◽

Yanan Liu ◽

...

Keyword(s):

Fatty Acids ◽

Amino Acid ◽

Milk Fat ◽

Milk Protein ◽

Volatile Fatty Acids ◽

Rumen Fluid ◽

Abundant Species ◽

Metagenomic Sequencing ◽

Amino Acid Synthesis ◽

Prevotella Species

The rumen contains abundant microorganisms that aid in the digestion of lignocellulosic feed and are associated with host phenotype traits. Cows with extremely high milk protein and fat percentages (HPF; n = 3) and low milk protein and fat percentages (LPF; n = 3) were selected from 4000 lactating Holstein cows under the same nutritional and management conditions. We found that the total concentration of volatile fatty acids, acetate, butyrate, and propionate in the rumen fluid was significantly higher in the HPF group than in the LPF group. Moreover, we identified 38 most abundant species displaying differential richness between the two groups, in which Prevotella accounted for 68.8% of the species, with the highest abundance in the HPF group. Functional annotation based on the Kyoto Encyclopedia of Gene and Genome (KEGG), evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), and Carbohydrate-Active enzymes (CAZy) databases showed that the significantly more abundant species in the HPF group are enriched in carbohydrate, amino acid, pyruvate, insulin, and lipid metabolism and transportation. Furthermore, Spearman’s rank correlation analysis revealed that specific microbial taxa (mainly the Prevotella species and Neocallimastix californiae) are positively correlated with total volatile fatty acids (VFA). Collectively, we found that the HPF group was enriched with several Prevotella species related to the total VFA, acetate, and amino acid synthesis. Thereby, these fulfilled the host’s needs for energy, fat, and rumen microbial protein, which can be used for increased biosynthesis of milk fat and milk protein. Our findings provide novel information for elucidation of the regulatory mechanism of the rumen in the formation of milk composition.

Download Full-text

Discriminating between JCPyV and BKPyV in Urinary Virome Data Sets

Viruses ◽

10.3390/v13061041 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1041

Author(s):

Rita Mormando ◽

Alan J. Wolfe ◽

Catherine Putonti

Keyword(s):

Jc Virus ◽

Sequence Similarity ◽

Bk Virus ◽

Data Sets ◽

Metagenomic Sequencing ◽

Significant Sequence Similarity ◽

Sequence Identity ◽

Shotgun Metagenomic Sequencing ◽

Urinary Microbiome ◽

Six Genes

Polyomaviruses are abundant in the human body. The polyomaviruses JC virus (JCPyV) and BK virus (BKPyV) are common viruses in the human urinary tract. Prior studies have estimated that JCPyV infects between 20 and 80% of adults and that BKPyV infects between 65 and 90% of individuals by age 10. However, these two viruses encode for the same six genes and share 75% nucleotide sequence identity across their genomes. While prior urinary virome studies have repeatedly reported the presence of JCPyV, we were interested in seeing how JCPyV prevalence compares to BKPyV. We retrieved all publicly available shotgun metagenomic sequencing reads from urinary microbiome and virome studies (n = 165). While one third of the data sets produced hits to JCPyV, upon further investigation were we able to determine that the majority of these were in fact BKPyV. This distinction was made by specifically mining for JCPyV and BKPyV and considering uniform coverage across the genome. This approach provides confidence in taxon calls, even between closely related viruses with significant sequence similarity.

Download Full-text

Metagenomic sequencing provides new insights into the subgingival bacteriome and aetiopathology of periodontitis

Journal of Periodontal Research ◽

10.1111/jre.12811 ◽

2021 ◽

Author(s):

Ethan Ng ◽

John R. H. Tay ◽

P. Balan ◽

Marianne M. A. Ong ◽

Nagihan Bostanci ◽

...

Keyword(s):

Metagenomic Sequencing

Download Full-text

Combined effects of composting and antibiotic administration on cattle manure–borne antibiotic resistance genes

Microbiome ◽

10.1186/s40168-021-01006-z ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Ishi Keenum ◽

Robert K. Williams ◽

Partha Ray ◽

Emily D. Garner ◽

Katharine F. Knowlton ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Land Application ◽

Multidimensional Analysis ◽

Small Scale ◽

Antibiotic Administration ◽

Metagenomic Sequencing ◽

Combined Effects ◽

Bacterial Microbiota

Abstract Background Research is needed to delineate the relative and combined effects of different antibiotic administration and manure management practices in either amplifying or attenuating the potential for antibiotic resistance to spread. Here, we carried out a comprehensive parallel examination of the effects of small-scale (> 55 °C × 3 days) static and turned composting of manures from dairy and beef cattle collected during standard antibiotic administration (cephapirin/pirlimycin or sulfamethazine/chlortetracycline/tylosin, respectively), versus from untreated cattle, on “resistomes” (total antibiotic resistance genes (ARGs) determined via shotgun metagenomic sequencing), bacterial microbiota, and indicator ARGs enumerated via quantitative polymerase chain reaction. To gain insight into the role of the thermophilic phase, compost was also externally heated to > 55 °C × 15 days. Results Progression of composting with time and succession of the corresponding bacterial microbiota was the overarching driver of the resistome composition (ANOSIM; R = 0.424, p = 0.001, respectively) in all composts at the small-scale. Reduction in relative abundance (16S rRNA gene normalized) of total ARGs in finished compost (day 42) versus day 0 was noted across all conditions (ANOSIM; R = 0.728, p = 0.001), except when externally heated. Sul1, intI1, beta-lactam ARGs, and plasmid-associated genes increased in all finished composts as compared with the initial condition. External heating more effectively reduced certain clinically relevant ARGs (blaOXA, blaCARB), fecal coliforms, and resistome risk scores, which take into account putative pathogen annotations. When manure was collected during antibiotic administration, taxonomic composition of the compost was distinct according to nonmetric multidimensional analysis and tet(W) decayed faster in the dairy manure with antibiotic condition and slower in the beef manure with antibiotic condition. Conclusions This comprehensive, integrated study revealed that composting had a dominant effect on corresponding resistome composition, while little difference was noted as a function of collecting manure during antibiotic administration. Reduction in total ARGs, tet(W), and resistome risk suggested that composting reduced some potential for antibiotic resistance to spread, but the increase and persistence of other indicators of antibiotic resistance were concerning. Results indicate that composting guidelines intended for pathogen reduction do not necessarily provide a comprehensive barrier to ARGs or their mobility prior to land application and additional mitigation measures should be considered.

Download Full-text

Characterizing and Evaluating the Zoonotic Potential of Novel Viruses Discovered in Vampire Bats

Viruses ◽

10.3390/v13020252 ◽

2021 ◽

Vol 13 (2) ◽

pp. 252

Author(s):

Laura M. Bergner ◽

Nardus Mollentze ◽

Richard J. Orton ◽

Carlos Tello ◽

Alice Broos ◽

...

Keyword(s):

Machine Learning ◽

Phylogenetic Analyses ◽

Human Infection ◽

Machine Learning Algorithms ◽

Zoonotic Potential ◽

Metagenomic Sequencing ◽

Learning Models ◽

Sequencing Data ◽

Vampire Bats ◽

Machine Learning Models

The contemporary surge in metagenomic sequencing has transformed knowledge of viral diversity in wildlife. However, evaluating which newly discovered viruses pose sufficient risk of infecting humans to merit detailed laboratory characterization and surveillance remains largely speculative. Machine learning algorithms have been developed to address this imbalance by ranking the relative likelihood of human infection based on viral genome sequences, but are not yet routinely applied to viruses at the time of their discovery. Here, we characterized viral genomes detected through metagenomic sequencing of feces and saliva from common vampire bats (Desmodus rotundus) and used these data as a case study in evaluating zoonotic potential using molecular sequencing data. Of 58 detected viral families, including 17 which infect mammals, the only known zoonosis detected was rabies virus; however, additional genomes were detected from the families Hepeviridae, Coronaviridae, Reoviridae, Astroviridae and Picornaviridae, all of which contain human-infecting species. In phylogenetic analyses, novel vampire bat viruses most frequently grouped with other bat viruses that are not currently known to infect humans. In agreement, machine learning models built from only phylogenetic information ranked all novel viruses similarly, yielding little insight into zoonotic potential. In contrast, genome composition-based machine learning models estimated different levels of zoonotic potential, even for closely related viruses, categorizing one out of four detected hepeviruses and two out of three picornaviruses as having high priority for further research. We highlight the value of evaluating zoonotic potential beyond ad hoc consideration of phylogeny and provide surveillance recommendations for novel viruses in a wildlife host which has frequent contact with humans and domestic animals.

Download Full-text