scholarly journals Rapid Diagnostics of Orthopaedic-Implant-Associated Infections Using Nanopore Shotgun Metagenomic Sequencing on Tissue Biopsies

2021 ◽  
Vol 9 (1) ◽  
pp. 97
Author(s):  
J. Christopher Noone ◽  
Karin Helmersen ◽  
Truls Michael Leegaard ◽  
Inge Skråmm ◽  
Hege Vangstein Aamot
Keyword(s):  
Antimicrobial Resistance ◽  
University Hospital ◽  
Orthopaedic Implant ◽  
Metagenomic Sequencing ◽  
Pathogen Identification ◽  
Shotgun Metagenomics ◽  
Microbial Identification ◽  
Conventional Culture ◽  
Shotgun Metagenomic Sequencing ◽  
Antimicrobial Resistance Gene

Conventional culture-based diagnostics of orthopaedic-implant-associated infections (OIAIs) are arduous. Hence, the aim of this study was to evaluate a culture-independent, rapid nanopore-based diagnostic protocol with regard to (a) pathogen identification, (b) time to pathogen identification, and (c) identification of antimicrobial resistance (AMR). This prospective proof-of-concept study included soft tissue biopsies from 32 patients with OIAIs undergoing first revision surgery at Akershus University Hospital, Norway. The biopsies were divided into two segments. Nanopore shotgun metagenomic sequencing and pathogen and antimicrobial resistance gene identification using the EPI2ME analysis platform (Oxford Nanopore Technologies) were performed on one segment. Conventional culture-based diagnostics were performed on the other. Microbial identification matched in 23/32 OIAI patients (72%). Sequencing detected additional microbes in 9/32 patients. Pathogens detected by culturing were identified by sequencing within a median of 1 h of sequencing start [range 1–18 h]. Phenotypic AMR was explained by the detection of resistance genes in 11/23 patients (48%). Diagnostics of OIAIs using shotgun metagenomics sequencing are possible within 24 h from biopsy using nanopore technology. Sequencing outperformed culturing with respect to speed and pathogen detection where pathogens were at sufficient concentration, whereas culture-based methods had an advantage at lower pathogen concentrations. Sequencing-based AMR detection may not yet be a suitable replacement for culture-based antibiotic susceptibility testing.

scholarly journals Culture-Independent Genotyping, Virulence and Antimicrobial Resistance Gene Identification of Staphylococcus aureus from Orthopaedic Implant-Associated Infections

2021 ◽  
Vol 9 (4) ◽  
pp. 707
Author(s):  
J. Christopher Noone ◽  
Fabienne Antunes Ferreira ◽  
Hege Vangstein Aamot
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Virulence Gene ◽  
Orthopaedic Implant ◽  
Metagenomic Sequencing ◽  
Gene Detection ◽  
Shotgun Metagenomic Sequencing ◽  
Antimicrobial Resistance Genes ◽  
Culture Independent

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.

scholarly journals Investigation of Spaceflight Induced Changes to Astronaut Microbiomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Michael D. Morrison ◽  
James B. Thissen ◽  
Fathi Karouia ◽  
Satish Mehta ◽  
Camilla Urbaniak ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
Relative Abundance ◽  
Human Microbiome ◽  
Metagenomic Sequencing ◽  
Beta Lactam ◽  
Gene Markers ◽  
Shotgun Metagenomic Sequencing ◽  
Antimicrobial Resistance Genes ◽  
Antimicrobial Resistance Gene

The International Space Station (ISS) is a uniquely enclosed environment that has been continuously occupied for the last two decades. Throughout its operation, protecting the health of the astronauts on-board has been a high priority. The human microbiome plays a significant role in maintaining human health, and disruptions in the microbiome have been linked to various diseases. To evaluate the effects of spaceflight on the human microbiome, body swabs and saliva samples were collected from four ISS astronauts on consecutive expeditions. Astronaut samples were analyzed using shotgun metagenomic sequencing and microarrays to characterize the microbial biodiversity before, during, and after the astronauts’ time onboard the ISS. Samples were evaluated at an individual and population level to identify changes in microbial diversity and abundance. No significant changes in the number or relative abundance of taxa were observed between collection time points when samples from all four astronauts were analyzed together. When the astronauts’ saliva samples were analyzed individually, the saliva samples of some astronauts showed significant changes in the relative abundance of taxa during and after spaceflight. The relative abundance of Prevotella in saliva samples increased during two astronauts’ time onboard the ISS while the relative abundance of other commensal taxa such as Neisseria, Rothia, and Haemophilus decreased. The abundance of some antimicrobial resistance genes within the saliva samples also showed significant changes. Most notably, elfamycin resistance gene significantly increased in all four astronauts post-flight and a CfxA6 beta-lactam marker significantly increased during spaceflight but returned to normal levels post-flight. The combination of both shotgun metagenomic sequencing and microarrays showed the benefit of both technologies in monitoring microbes on board the ISS. There were some changes in each astronaut’s microbiome during spaceflight, but these changes were not universal for all four astronauts. Two antimicrobial resistance gene markers did show a significant change in abundance in the saliva samples of all four astronauts across their collection times. These results provide insight for future ISS microbial monitoring studies and targets for antimicrobial resistance screenings.

scholarly journals Diversity and Function of Wolf Spider Gut Microbiota Revealed by Shotgun Metagenomics

2021 ◽  
Vol 12 ◽  
Author(s):  
Runbiao Wu ◽  
Luyu Wang ◽  
Jianping Xie ◽  
Zhisheng Zhang
Keyword(s):  
Gut Microbiota ◽  
Wolf Spider ◽  
Metagenomic Sequencing ◽  
Wolf Spiders ◽  
Shotgun Metagenomics ◽  
Microbial Genomes ◽  
Shotgun Metagenomic Sequencing ◽  
Crucial Component ◽  
Genes Encoding ◽  
And Function

Wolf spiders (Lycosidae) are crucial component of integrated pest management programs and the characteristics of their gut microbiota are known to play important roles in improving fitness and survival of the host. However, there are only few studies of the gut microbiota among closely related species of wolf spider. Whether wolf spiders gut microbiota vary with habitats remains unknown. Here, we used shotgun metagenomic sequencing to compare the gut microbiota of two wolf spider species, Pardosa agraria and P. laura from farmland and woodland ecosystems, respectively. The results show that the gut microbiota of Pardosa spiders is similar in richness and abundance. Approximately 27.3% of the gut microbiota of P. agraria comprises Proteobacteria, and approximately 34.5% of the gut microbiota of P. laura comprises Firmicutes. We assembled microbial genomes and found that the gut microbiota of P. laura are enriched in genes for carbohydrate metabolism. In contrast, those of P. agraria showed a higher proportion of genes encoding acetyltransferase, an enzyme involved in resistance to antibiotics. We reconstructed three high-quality and species-level microbial genomes: Vulcaniibacterium thermophilum, Anoxybacillus flavithermus and an unknown bacterium belonging to the family Simkaniaceae. Our results contribute to an understanding of the diversity and function of gut microbiota in closely related spiders.

scholarly journals Mesophilic Sporeformers Identified in Whey Powder by Using Shotgun Metagenomic Sequencing

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Aoife J. McHugh ◽  
Conor Feehily ◽  
John T. Tobin ◽  
Mark A. Fenelon ◽  
Colin Hill ◽  
...  
Keyword(s):  
Dairy Product ◽  
Metagenomic Sequencing ◽  
Shotgun Metagenomics ◽  
Food Ingredients ◽  
Content Type ◽  
Whey Powder ◽  
Shotgun Metagenomic Sequencing ◽  
Spoilage Bacteria ◽  
Culture Independent ◽  
Spore Forming Bacteria

ABSTRACT Spoilage and pathogenic spore-forming bacteria are a major cause of concern for producers of dairy products. Traditional agar-based detection methods employed by the dairy industry have limitations with respect to their sensitivity and specificity. The aim of this study was to identify low-abundance sporeformers in samples of a powdered dairy product, whey powder, produced monthly over 1 year, using novel culture-independent shotgun metagenomics-based approaches. Although mesophilic sporeformers were the main target of this study, in one instance thermophilic sporeformers were also targeted using this culture-independent approach. For comparative purposes, mesophilic and thermophilic sporeformers were also tested for within the same sample using culture-based approaches. Ultimately, the approaches taken highlighted differences in the taxa identified due to treatment and isolation methods. Despite this, low levels of transient, mesophilic, and in some cases potentially pathogenic sporeformers were consistently detected in powder samples. Although the specific sporeformers changed from one month to the next, it was apparent that 3 groups of mesophilic sporeformers, namely, Bacillus cereus, Bacillus licheniformis/Bacillus paralicheniformis, and a third, more heterogeneous group containing Brevibacillus brevis, dominated across the 12 samples. Total thermophilic sporeformer taxonomy was considerably different from mesophilic taxonomy, as well as from the culturable thermophilic taxonomy, in the one sample analyzed by all four approaches. Ultimately, through the application of shotgun metagenomic sequencing to dairy powders, the potential for this technology to facilitate the detection of undesirable bacteria present in these food ingredients is highlighted. IMPORTANCE The ability of sporeformers to remain dormant in a desiccated state is of concern from a safety and spoilage perspective in dairy powder. Traditional culturing techniques are slow and provide little information without further investigation. We describe the identification of mesophilic sporeformers present in powders produced over 1 year, using novel shotgun metagenomic sequencing. This method allows detection and identification of possible pathogens and spoilage bacteria in parallel. Strain-level analysis and functional gene analysis, such as identification of toxin genes, were also performed. This approach has the potential to be of great value with respect to the detection of spore-forming bacteria and could allow a processor to make an informed decision surrounding process changes to reduce the risk of spore contamination.

scholarly journals The unintended consequences of a surge in antibiotic resistance when removing wastewater drain biofilm

2020 ◽  
Author(s):  
Shireen M Kotay ◽  
Hardik I Parikh ◽  
Hyun S Gweon ◽  
Katie Barry ◽  
Nicole Stoesser ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Pathogenic Bacteria ◽  
Biotic Interactions ◽  
Care Patient ◽  
Unintended Consequences ◽  
Resistant Bacteria ◽  
Microbial Culture ◽  
Metagenomic Sequencing ◽  
Shotgun Metagenomic Sequencing ◽  
Critical Issues

SUMMARYThe prevalence and proliferation of antimicrobial resistant bacteria is considered one of the critical issues of our time. Wastewater is a habitat for complex microbial communities where bacteria share genes of antimicrobial resistance through horizontal gene transfer. Hospital wastewater drainage systems are an ideal reservoir for environmental and pathogenic bacteria to interface and exchange genes of antimicrobial resistance1. Highly resistant Enterobacterales are of the greatest concern because they not only cause human infections but can also thrive in the environment2,3 and have shown to transmit from sink drains to patients4. Replacement of contaminated plumbing may be the most intuitive and widely deployed response once highly resistant potentially pathogenic bacteria are identified in a sink drain. Here we show using shotgun metagenomic sequencing of sink drain biofilms (in 6 intensive care patient rooms, 36 subsamples) paired with microbial culture analysis, an evident shift of biofilm community structure towards increased abundance of Enterobacteriaceae in the newly replaced sink plumbing. Significant increase in resistome load and abundance of clinically relevant resistance and typically mobile genes in the newly replaced plumbing was also observed. Findings from this study demonstrate that exchanging contaminated plumbing for new plumbing may actually have the unexpected consequence of increased abundance of Enterobacterales and genes of antimicrobial resistance in the sink drains. Disruption of preexisting complex environmental biofilms may result in an unintended increase in the amount of antimicrobial resistant Enterobacterales which are targeted for elimination. Temporal variability is an important attribute of ecosystems affecting colonization dynamics and biotic interactions (composition of introduced and resident communities) are key to functional outcomes. Absence of competition within an ecosystem favors invasion.

Shotgun metagenomics reveals both taxonomic and tryptophan pathway differences of gut microbiota in major depressive disorder patients

2019 ◽  
pp. 1-12 ◽  
Author(s):  
Wen-tao Lai ◽  
Wen-feng Deng ◽  
Shu-xian Xu ◽  
Jie Zhao ◽  
Dan Xu ◽  
...  
Keyword(s):  
Major Depressive Disorder ◽  
Gut Microbiota ◽  
Depressive Disorder ◽  
Characteristic Curve ◽  
Critical Role ◽  
Metagenomic Sequencing ◽  
Major Depressive ◽  
Shotgun Metagenomics ◽  
Shotgun Metagenomic Sequencing ◽  
Tryptophan Pathway

Abstract Background The microbiota–gut–brain axis, especially the microbial tryptophan (Trp) biosynthesis and metabolism pathway (MiTBamp), may play a critical role in the pathogenesis of major depressive disorder (MDD). However, studies on the MiTBamp in MDD are lacking. The aim of the present study was to analyze the gut microbiota composition and the MiTBamp in MDD patients. Methods We performed shotgun metagenomic sequencing of stool samples from 26 MDD patients and 29 healthy controls (HCs). In addition to the microbiota community and the MiTBamp analyses, we also built a classification based on the Random Forests (RF) and Boruta algorithm to identify the gut microbiota as biomarkers for MDD. Results The Bacteroidetes abundance was strongly reduced whereas that of Actinobacteria was significantly increased in the MDD patients compared with the abundance in the HCs. Most noteworthy, the MDD patients had increased levels of Bifidobacterium, which is commonly used as a probiotic. Four Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologies (KOs) (K01817, K11358, K01626, K01667) abundances in the MiTBamp were significantly lower in the MDD group. Furthermore, we found a negative correlation between the K01626 abundance and the HAMD scores in the MDD group. Finally, RF classification at the genus level can achieve an area under the receiver operating characteristic curve of 0.890. Conclusions The present findings enabled a better understanding of the changes in gut microbiota and the related Trp pathway in MDD. Alterations of the gut microbiota may have the potential as biomarkers for distinguishing MDD patients form HCs.

scholarly journals ResistoXplorer: a web-based tool for visual, statistical and exploratory data analysis of resistome data

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Achal Dhariwal ◽  
Roger Junges ◽  
Tsute Chen ◽  
Fernanda C Petersen
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Gene ◽  
High Throughput ◽  
Resistance Genes ◽  
Visual Analytics ◽  
Gene List ◽  
Metagenomic Sequencing ◽  
High Throughput Analysis ◽  
Antimicrobial Resistance Genes ◽  
Antimicrobial Resistance Gene

Abstract The study of resistomes using whole metagenomic sequencing enables high-throughput identification of resistance genes in complex microbial communities, such as the human microbiome. Over recent years, sophisticated and diverse pipelines have been established to facilitate raw data processing and annotation. Despite the progress, there are no easy-to-use tools for comprehensive visual, statistical and functional analysis of resistome data. Thus, exploration of the resulting large complex datasets remains a key bottleneck requiring robust computational resources and technical expertise, which creates a significant hurdle for advancements in the field. Here, we introduce ResistoXplorer, a user-friendly tool that integrates recent advancements in statistics and visualization, coupled with extensive functional annotations and phenotype collection, to enable high-throughput analysis of common outputs generated from metagenomic resistome studies. ResistoXplorer contains three modules—the ‘Antimicrobial Resistance Gene Table’ module offers various options for composition profiling, functional profiling and comparative analysis of resistome data; the ‘Integration’ module supports integrative exploratory analysis of resistome and microbiome abundance profiles derived from metagenomic samples; finally, the ‘Antimicrobial Resistance Gene List’ module enables users to intuitively explore the associations between antimicrobial resistance genes and the microbial hosts using network visual analytics to gain biological insights. ResistoXplorer is publicly available at http://www.resistoxplorer.no.

scholarly journals Applying Genome-Resolved Metagenomics to Deconvolute the Halophilic Microbiome

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 220 ◽  
Author(s):  
Gherman Uritskiy ◽  
Jocelyne DiRuggiero
Keyword(s):  
Microbial Communities ◽  
Intraspecific Diversity ◽  
Metagenomic Sequencing ◽  
Chromosome Conformation ◽  
Shotgun Metagenomics ◽  
Sequencing Technologies ◽  
Shotgun Metagenomic Sequencing ◽  
Long Read ◽  
Analytical Strategies ◽  
Potential Applications

In the past decades, the study of microbial life through shotgun metagenomic sequencing has rapidly expanded our understanding of environmental, synthetic, and clinical microbial communities. Here, we review how shotgun metagenomics has affected the field of halophilic microbial ecology, including functional potential reconstruction, virus–host interactions, pathway selection, strain dispersal, and novel genome discoveries. However, there still remain pitfalls and limitations from conventional metagenomic analysis being applied to halophilic microbial communities. Deconvolution of halophilic metagenomes has been difficult due to the high G + C content of these microbiomes and their high intraspecific diversity, which has made both metagenomic assembly and binning a challenge. Halophiles are also underrepresented in public genome databases, which in turn slows progress. With this in mind, this review proposes experimental and analytical strategies to overcome the challenges specific to the halophilic microbiome, from experimental designs to data acquisition and the computational analysis of metagenomic sequences. Finally, we speculate about the potential applications of other next-generation sequencing technologies in halophilic communities. RNA sequencing, long-read technologies, and chromosome conformation assays, not initially intended for microbiomes, are becoming available in the study of microbial communities. Together with recent analytical advancements, these new methods and technologies have the potential to rapidly advance the field of halophile research.

scholarly journals Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens

2019 ◽  
Vol 5 (3) ◽  
pp. 430-442 ◽  
Author(s):  
Richard M. Leggett ◽  
Cristina Alcon-Giner ◽  
Darren Heavens ◽  
Shabhonam Caim ◽  
Thomas C. Brook ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Real Time ◽  
Software Package ◽  
Pathogenic Bacteria ◽  
Clinical Samples ◽  
Clinical Settings ◽  
Metagenomic Sequencing ◽  
Antimicrobial Resistance Genes ◽  
Antimicrobial Resistance Gene ◽  
Mock Communities

AbstractThe MinION sequencing platform offers near real-time analysis of DNA sequence; this makes the tool attractive for deployment in fieldwork or clinical settings. We used the MinION platform coupled to the NanoOK RT software package to perform shotgun metagenomic sequencing and profile mock communities and faecal samples from healthy and ill preterm infants. Using Nanopore data, we reliably classified a 20-species mock community and captured the diversity of the immature gut microbiota over time and in response to interventions such as probiotic supplementation, antibiotic treatment or episodes of suspected sepsis. We also performed rapid real-time runs to assess gut-associated microbial communities in critically ill and healthy infants, facilitated by NanoOK RT software package, which analysed sequences as they were generated. Our pipeline reliably identified pathogenic bacteria (that is, Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance gene profiles within as little as 1 h of sequencing. Results were confirmed using pathogen isolation, whole-genome sequencing and antibiotic susceptibility testing, as well as mock communities and clinical samples with known antimicrobial resistance genes. Our results demonstrate that MinION (including cost-effective Flongle flow cells) with NanoOK RT can process metagenomic samples to a rich dataset in < 5 h, which creates a platform for future studies aimed at developing these tools and approaches in clinical settings with a focus on providing tailored patient antimicrobial treatment options.

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