scholarly journals Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

Water ◽  
2021 ◽  
Vol 13 (18) ◽  
pp. 2503
Author(s):  
Giuseppina La Rosa ◽  
David Brandtner ◽  
Pamela Mancini ◽  
Carolina Veneri ◽  
Giusy Bonanno Ferraro ◽  
...  
Keyword(s):  
Central Italy ◽  
Amplicon Sequencing ◽  
Wastewater Sample ◽  
Global Public Health ◽  
Sample Collection ◽  
Increased Risk ◽  
Long Read ◽  
Alpha Variant ◽  
Wastewater Samples ◽  
Alpha Cluster

The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) poses an increased risk to global public health and underlines the need to prioritise monitoring and research to better respond to the COVID-19 pandemic. Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. In conclusion, wastewater monitoring can help one discover the spread of variants in a community and early detect the emerging of clinically relevant mutations or variants.

scholarly journals Molecular Epidemiology of SARS-CoV-2 in Diverse Environmental Samples Globally

2021 ◽  
Vol 9 (8) ◽  
pp. 1696
Author(s):  
Ariful Islam ◽  
Md. Abu Sayeed ◽  
Md. Abul Kalam ◽  
Jinnat Ferdous ◽  
Md. Kaisar Rahman ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Human Population ◽  
Virus Genome ◽  
Global Environment ◽  
Amino Acid Substitutions ◽  
Wastewater Sample ◽  
Environmental Surveillance ◽  
Alpha Variant ◽  
Wastewater Samples ◽  
Proper Disposal

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swamped the global environment greatly in the current pandemic. Wastewater-based epidemiology (WBE) effectively forecasts the surge of COVID-19 cases in humans in a particular region. To understand the genomic characteristics/footprints and diversity of SARS-CoV-2 in the environment, we analyzed 807 SARS-CoV-2 sequences from 20 countries deposited in GISAID till 22 May 2021. The highest number of sequences (n = 638) were reported in Austria, followed by the Netherlands, China, and Bangladesh. Wastewater samples were highest (40.0%) to successfully yield the virus genome followed by a 24 h composite wastewater sample (32.6%) and sewage (18.5%). Phylogenetic analysis revealed that SARS-CoV-2 environmental strains are a close congener with the strains mostly circulating in the human population from the same region. Clade GRY (32.7%), G (29.2%), GR (25.3%), O (7.2%), GH (3.4%), GV (1.4%), S (0.5%), and L (0.4%) were found in environmental samples. Various lineages were identified in environmental samples; nevertheless, the highest percentages (49.4%) of the alpha variant (B.1.1.7) were detected in Austria, Liechtenstein, Slovenia, Czech Republic, Switzerland, Germany, and Italy. Other prevalent lineages were B.1 (18.2%), B.1.1 (9.2%), and B.1.160 (3.9%). Furthermore, a significant number of amino acid substitutions were found in environmental strains where the D614G was found in 83.8% of the sequences. However, the key mutations—N501Y (44.6%), S982A (44.4%), A570D (43.3%), T716I (40.4%), and P681H (40.1%) were also recorded in spike protein. The identification of the environmental belvedere of SARS-CoV-2 and its genetic signature is crucial to detect outbreaks, forecast pandemic harshness, and prepare with the appropriate tools to control any impending pandemic. We recommend genomic environmental surveillance to trace the emerging variants and diversity of SARS-CoV-2 viruses circulating in the community. Additionally, proper disposal and treatment of wastewater, sewage, and medical wastes are important to prevent environmental contamination.

scholarly journals Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Benjamin J. Callahan ◽  
Dmitry Grinevich ◽  
Siddhartha Thakur ◽  
Michael A. Balamotis ◽  
Tuval Ben Yehezkel
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Strain Identification ◽  
Long Reads ◽  
Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

scholarly journals Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Caitlin M. Singleton ◽  
Francesca Petriglieri ◽  
Jannie M. Kristensen ◽  
Rasmus H. Kirkegaard ◽  
Thomas Y. Michaelsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Sequencing Data ◽  
High Quality ◽  
16S Rrna Amplicon Sequencing ◽  
Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

scholarly journals Long-Read Amplicon Sequencing of Nitric Oxide Dismutase (nod) Genes Reveal Diverse Oxygenic Denitrifiers in Agricultural Soils and Lake Sediments

2020 ◽  
Vol 80 (1) ◽  
pp. 243-247 ◽  
Author(s):  
Baoli Zhu ◽  
Zhe Wang ◽  
Dheeraj Kanaparthi ◽  
Susanne Kublik ◽  
Tida Ge ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Lake Sediments ◽  
Agricultural Soils ◽  
Amplicon Sequencing ◽  
Nod Genes ◽  
Long Read

scholarly journals Feasibility and acceptability of a universal screening and referral protocol for gender-based violence with women seeking care in health clinics in Dadaab refugee camps in Kenya

2017 ◽  
Vol 4 ◽  
Author(s):  
A. Vu ◽  
A. L. Wirtz ◽  
S. Bundgaard ◽  
A. Nair ◽  
G. Luttah ◽  
...  
Keyword(s):  
Universal Screening ◽  
Service Providers ◽  
Public Health Problem ◽  
Global Public Health ◽  
Gender Based Violence ◽  
Internally Displaced ◽  
Health Clinics ◽  
Increased Risk ◽  
Screening Protocol ◽  
Gender Based

Background.Gender-based violence (GBV) is both a global public health problem and violation of human rights. Refugees and internally displaced persons experience an increased risk of GBV and health outcomes associated with GBV are often exacerbated in conflict settings.Methods.A mixed methods study to examine the feasibility and acceptability of universal screening for GBV in a refugee population in the Dadaab refugee camp of Kenya, using the ASIST-GBV from January to July 2015.Results.Of 9366 women offered screening at International Rescue Committee health clinics, about 89% (n = 8369) female refugees consented to participate. Only 15% of the potentially eligible population could participate in GBV screening because of the ongoing struggle to identify private space in the clinics. Over 85% of women reported being ‘willing’ or ‘very willing’ to participate in GBV screening; 96% felt they had a good or very good experience with the screening protocol. Qualitative findings stressed the importance of securing a room/space in the busy clinic is critical to universal screening with referral to safe and confidential services for survivors.Conclusions.The findings suggest that the evidence-based ASIST-GBV is both feasible to implement and acceptable to both providers and women seeking care. Universal GBV screening and referral is an effective way for health care and service providers in humanitarian settings to assist survivors of GBV.

scholarly journals 16S rRNA Long-Read Sequencing of the Granulation Tissue from Nonsmokers and Smokers-Severe Chronic Periodontitis Patients

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Rebecca Chowdhry ◽  
Neetu Singh ◽  
Dinesh Kumar Sahu ◽  
Ratnesh Kumar Tripathi ◽  
Archana Mishra ◽  
...  
Keyword(s):  
16S Rrna ◽  
Granulation Tissue ◽  
Ketone Bodies ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Differential Abundance ◽  
Streptobacillus Moniliformis ◽  
Increased Risk ◽  
Long Read ◽  
Significant Enrichment

Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity among smokers and nonsmokers which may help in better understanding of predisposition to this chronic inflammation mediated diseases. We selected deep-seated infected granulation tissue removed during periodontal flap surgery procedures for identification and differential abundance of residential bacterial species among smokers and nonsmokers through long-read sequencing technology targeting full-length 16S rRNA gene. A total of 8 phyla were identified among which Firmicutes and Bacteroidetes were most dominating. Differential abundance analysis of OTUs through PICRUST showed significant (p>0.05) abundance of Phyla-Fusobacteria (Streptobacillus moniliformis); Phyla-Firmicutes (Streptococcus equi), and Phyla Proteobacteria (Enhydrobacter aerosaccus) in nonsmokers compared to smokers. The differential abundance of oral metagenomes in smokers showed significant enrichment of host genes modulating pathways involving primary immunodeficiency, citrate cycle, streptomycin biosynthesis, vitamin B6 metabolism, butanoate metabolism, glycine, serine, and threonine metabolism pathways. While thiamine metabolism, amino acid metabolism, homologous recombination, epithelial cell signaling, aminoacyl-tRNA biosynthesis, phosphonate/phosphinate metabolism, polycyclic aromatic hydrocarbon degradation, synthesis and degradation of ketone bodies, translation factors, Ascorbate and aldarate metabolism, and DNA replication pathways were significantly enriched in nonsmokers, modulation of these pathways in oral cavities due to differential enrichment of metagenomes in smokers may lead to an increased susceptibility to infections and/or higher formation of DNA adducts, which may increase the risk of carcinogenesis.

scholarly journals COPD, Depression and COVID-19 Pandemic: A Harrowing, Demoralizing and Deleterious Triad

2021 ◽  
Vol 3 (8) ◽  
pp. 01-06
Author(s):  
Iqbal Akhtar Khan ◽  
Hamza Iltaf Malik
Keyword(s):  
Hospital Admissions ◽  
Hospital Readmissions ◽  
Public Health Problem ◽  
Well Being ◽  
Care Utilization ◽  
Global Public Health ◽  
Increased Risk ◽  
Co Morbidity ◽  
Pulmonary Manifestations ◽  
The Impact

COPD is a highly incapacitating global public health problem, with pulmonary and extra-pulmonary manifestations and usually associated with significant concomitant chronic diseases. With enhanced understanding, it has extensively been reported as a complex, heterogeneous and dynamic disease affecting patients’ health beyond pulmones. Depression, with prevalence of 322 million people, is a major contributor to the overall global burden of disease. In various epidemiological and clinical studies, its prevalence among patients with COPD varies from 18% to 80%. This deadly duo leads to excessive health care utilization rates and costs including increased rates of exacerbation, sub-optimal adherence to prescribed medications, increased hospital admissions, longer hospital stays and increased hospital readmissions. Moreover, there is increased risk of suicidal ideation, suicidal attempts, and suicidal drug overdose. It is a pity that, in significant cases, the co-morbidity remains under-recognized and under-treated. The impact of prevailing COVID 19 pandemic, on the dual burden of COPD and depression, and possible remedial measures including “The 6 ways to boost one’s well-being-by Mental Heath UK, “The Living with the Times” toolkit--by WHO” and innovative add-ons like Dance Movement Therapy and Musical Engagement Therapy have been discussed.

scholarly journals Dynamics of blaKPC-2 Dissemination from Non-CG258 Klebsiella pneumoniae to Other Enterobacterales via IncN Plasmids in an Area of High Endemicity

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Ana M. Rada ◽  
Elsa De La Cadena ◽  
Carlos Agudelo ◽  
Cesar Capataz ◽  
Nataly Orozco ◽  
...  
Keyword(s):  
Public Health ◽  
Klebsiella Pneumoniae ◽  
Neonatal Intensive Care ◽  
Carbapenem Resistance ◽  
Health Interventions ◽  
Global Public Health ◽  
Public Health Interventions ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Long Read

ABSTRACT Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of blaKPC dissemination have been reported in Colombia, a country with a high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of blaKPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015). We identified the genomic characteristics of KPC-producing Enterobacterales recovered from patients infected/colonized and reconstructed the dynamics of dissemination of blaKPC-2 using both short and long read sequencing. We found that spread of blaKPC-2 among Enterobacterales in the participating hospitals was due to intra- and interspecies horizontal gene transfer (HGT) mediated by promiscuous plasmids associated with transposable elements that was originated from a multispecies outbreak of KPC-producing Enterobacterales in a neonatal intensive care unit. The plasmids were detected in isolates recovered in other units within the same hospital and nearby hospitals. The gene “epidemic” was driven by IncN-pST15-type plasmids carrying a novel Tn4401b structure and non-Tn4401 elements (NTEKPC) in Klebsiella spp., Escherichia coli, Enterobacter spp., and Citrobacter spp. Of note, mcr-9 was found to coexist with blaKPC-2 in species of the Enterobacter cloacae complex. Our findings suggest that the main mechanism for dissemination of blaKPC-2 is HGT mediated by highly transferable plasmids among species of Enterobacterales in infected/colonized patients, presenting a major challenge for public health interventions in developing countries such as Colombia.

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