scholarly journals Getting close to nature – Plasmodium knowlesi reference genome sequences from contemporary clinical isolates

2021 ◽  
Author(s):  
Damilola R Oresegun ◽  
Peter Thorpe ◽  
Ernest Diez Benavente ◽  
Susana Campino ◽  
Muh Fauzi ◽  
...  
Keyword(s):  
Plasmodium Knowlesi ◽  
Sequence Data ◽  
Family Members ◽  
Gene Families ◽  
Core Gene ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Cell Agglutination ◽  
Sequencing Platform ◽  
Kir Genes

Plasmodium knowlesi, a malaria parasite of old-world macaque monkeys, is used extensively to model Plasmodium biology. Recently P. knowlesi was found in the human population of Southeast Asia, particularly Malaysia. P. knowlesi causes un-complicated to severe and fatal malaria in the human host with features in common with the more prevalent and virulent malaria caused by Plasmodium falciparum. As such P. knowlesi presents a unique opportunity to inform an experimental model for malaria with clinical data from same-species human infections. Experimental lines of P. knowlesi represent well characterised genetically static parasites and to maximise their utility as a backdrop for understanding malaria pathophysiology, genetically diverse contemporary clinical isolates, essentially wild-type, require comparable characterization. The Oxford Nanopore PCR-free long-read sequencing platform was used to sequence P. knowlesi parasites from archived clinical samples. The sequencing platform and assembly pipeline was designed to facilitate capturing data on important multiple gene families, including the P. knowlesi schizont-infected cell agglutination (SICA) var genes and the Knowlesi-Interspersed Repeats (KIR) genes. The SICAvar and KIR gene families code for antigenically variant proteins that have been difficult to resolve and characterise. Analyses presented here suggest that the family members have arisen through a process of gene duplication, selection pressure and variation. Highly evolving genes tend to be located proximal to genetic elements that drive change rather than regions that support core gene conservation. For example, the virulence-associated P. falciparum erythrocyte membrane protein (PfEMP1) gene family members are restricted to relatively unstable sub-telomeric regions. In contrast the SICAvar nd KIR genes are located throughout the genome but as the study presented here shows, they occupy otherwise gene-sparse chromosomal locations. The novel methods presented here offer the malaria research community new tools to generate comprehensive genome sequence data from small clinical samples and renewed insight into these complex real-world parasites.

scholarly journals Plasmodium knowlesi – Clinical Isolate Genome Sequencing to Inform Translational Same-Species Model System for Severe Malaria

2021 ◽  
Vol 11 ◽  
Author(s):  
Damilola R. Oresegun ◽  
Cyrus Daneshvar ◽  
Janet Cox-Singh
Keyword(s):  
Severe Malaria ◽  
Plasmodium Knowlesi ◽  
Sequence Data ◽  
Severe Disease ◽  
Model System ◽  
Clinical Samples ◽  
Laboratory Model ◽  
High Morbidity ◽  
Parasite Diversity ◽  
Sub Saharan

Malaria is responsible for unacceptably high morbidity and mortality, especially in Sub-Saharan African Nations. Malaria is caused by member species’ of the genus Plasmodium and despite concerted and at times valiant efforts, the underlying pathophysiological processes leading to severe disease are poorly understood. Here we describe zoonotic malaria caused by Plasmodium knowlesi and the utility of this parasite as a model system for severe malaria. We present a method to generate long-read third-generation Plasmodium genome sequence data from archived clinical samples using the MinION platform. The method and technology are accessible, affordable and data is generated in real-time. We propose that by widely adopting this methodology important information on clinically relevant parasite diversity, including multiple gene family members, from geographically distinct study sites will emerge. Our goal, over time, is to exploit the duality of P. knowlesi as a well-used laboratory model and human pathogen to develop a representative translational model system for severe malaria that is informed by clinically relevant parasite diversity.

scholarly journals Long read assemblies of geographically dispersed Plasmodium falciparum isolates reveal highly structured subtelomeres

2018 ◽  
Vol 3 ◽  
pp. 52 ◽  
Author(s):  
Thomas D. Otto ◽  
Ulrike Böhme ◽  
Mandy Sanders ◽  
Adam J. Reid ◽  
Ellen I. Bruske ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Sequence Data ◽  
Gene Families ◽  
Complete Sequence ◽  
Clinical Isolates ◽  
Current Standard ◽  
Entire Genome ◽  
Variable Regions ◽  
Long Read ◽  
Subtelomeric Regions

Background: Although thousands of clinical isolates of Plasmodium falciparum are being sequenced and analysed by short read technology, the data do not resolve the highly variable subtelomeric regions of the genomes that contain polymorphic gene families involved in immune evasion and pathogenesis. There is also no current standard definition of the boundaries of these variable subtelomeric regions. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated the genomes of 15 P. falciparum isolates, ten of which are newly cultured clinical isolates. We performed comparative analysis of the entire genome with particular emphasis on the subtelomeric regions and the internal var genes clusters.  Results: The nearly complete sequence of these 15 isolates has enabled us to define a highly conserved core genome, to delineate the boundaries of the subtelomeric regions, and to compare these across isolates. We found highly structured variable regions in the genome. Some exported gene families purportedly involved in release of merozoites show copy number variation. As an example of ongoing genome evolution, we found a novel CLAG gene in six isolates.  We also found a novel gene that was relatively enriched in the South East Asian isolates compared to those from Africa. Conclusions: These 15 manually curated new reference genome sequences with their nearly complete subtelomeric regions and fully assembled genes are an important new resource for the malaria research community. We report the overall conserved structure and pattern of important gene families and the more clearly defined subtelomeric regions.

scholarly journals WeFaceNano: a user-friendly pipeline for complete ONT sequence assembly and detection of antibiotic resistance in multi-plasmid bacterial isolates

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Astrid P. Heikema ◽  
Rick Jansen ◽  
Saskia D. Hiltemann ◽  
John P. Hays ◽  
Andrew P. Stubbs
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Dna Sequences ◽  
Sequence Data ◽  
Antibiotic Resistance Genes ◽  
Clinical Samples ◽  
Microbial Resistance ◽  
Sequencing Platform ◽  
Long Read ◽  
User Friendly

Abstract Background Bacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance. The ability to completely assemble plasmid sequences would facilitate the localization of antibiotic resistance genes, the identification of genes that promote plasmid transmission and the accurate tracking of plasmid mobility. However, the complete assembly of plasmid sequences using the currently most widely used sequencing platform (Illumina-based sequencing) is restricted due to the generation of short sequence lengths. The long-read Oxford Nanopore Technologies (ONT) sequencing platform overcomes this limitation. Still, the assembly of plasmid sequence data remains challenging due to software incompatibility with long-reads and the error rate generated using ONT sequencing. Bioinformatics pipelines have been developed for ONT-generated sequencing but require computational skills that frequently are beyond the abilities of scientific researchers. To overcome this challenge, the authors developed ‘WeFaceNano’, a user-friendly Web interFace for rapid assembly and analysis of plasmid DNA sequences generated using the ONT platform. WeFaceNano includes: a read statistics report; two assemblers (Miniasm and Flye); BLAST searching; the detection of antibiotic resistance- and replicon genes and several plasmid visualizations. A user-friendly interface displays the main features of WeFaceNano and gives access to the analysis tools. Results Publicly available ONT sequence data of 21 plasmids were used to validate WeFaceNano, with plasmid assemblages and anti-microbial resistance gene detection being concordant with the published results. Interestingly, the “Flye” assembler with “meta” settings generated the most complete plasmids. Conclusions WeFaceNano is a user-friendly open-source software pipeline suitable for accurate plasmid assembly and the detection of anti-microbial resistance genes in (clinical) samples where multiple plasmids can be present.

scholarly journals Plasmodium knowlesi clinical isolates from Malaysia show extensive diversity and strong differential selection pressure at the merozoite surface protein 7D (MSP7D)

2019 ◽  
Author(s):  
Md Atique Ahmed ◽  
Fu-Shi Quan
Keyword(s):  
Natural Selection ◽  
Central Region ◽  
Nucleotide Diversity ◽  
Plasmodium Knowlesi ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Full Length ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Geographical Clustering

AbstractBackgroundHigh proportion of human cases due to the simian malaria parasite Plasmodium knowlesi in Malaysia has been a cause of concern, as it can be severe and fatal. Merozoite surface protein 7 (MSP7) is a multigene family which forms a non-covalent complex with MSP-1 prior to receptor-ligand recognition in Plasmodium falciparum and thus an important antigen for vaccine development. However, no study has been done in any of the ortholog family members in Plasmodium knowlesi from clinical samples. Thus in this study we investigated the level of polymorphism, haplotypes, and natural selection acting at the pkmsp-7D gene in clinical samples from Malaysia.MethodsWe analyzed 36 full-length pkmsp7D gene sequences (along with the reference H-strain: PKNH_1266000) which were orthologous to pvmsp7H (PVX_082680) from clinical isolates of Malaysia available from public databases. Population genetic, evolutionary and phylogenetic analyses were performed to determine the level of genetic diversity, polymorphism, recombination and natural selection.ResultsAnalysis of 36 full-length pkmsp7D sequences identified 147 SNPs (91 non-synonymous and 56 synonymous substitutions). Nucleotide diversity across the full-length gene was higher than its ortholog in P. vivax (msp7H). Region-wise analysis of the gene indicated that the nucleotide diversity at the central region was very high (π = 0.14) compared to the 5’ and 3’ regions. Most hyper-variable SNPs were detected at the central domain. Multiple test for natural selection indicated the central region was under strong positive natural selection however, the 5’ and 3’ regions were under negative/purifying selection. Evidence of intragenic recombination were detected at the central region of the gene. Phylogenetic analysis using full-length msp7D genes indicated there was no geographical clustering of parasite population.ConclusionsHigh genetic diversity with hyper-variable SNPs and strong evidence of positive natural selection at the central region of MSP7D indicated exposure of the region to host immune pressure. Negative selection at the 5’ and the 3’ regions of MSP7D might be because of functional constraints at the unexposed regions during the merozoite invasion process of P. knowlesi. No evidence of geographical clustering among the clinical isolates from Malaysia indicated uniform selection pressure in all populations. These findings highlight the further evaluation of the regions and functional characterization of the protein as a potential blood stage vaccine candidate for P. knowlesi.

scholarly journals Approaches to estimating inbreeding coefficients in clinical isolates of Plasmodium falciparum from genomic sequence data

2015 ◽  
Author(s):  
Lucas Amenga-Etego ◽  
Ruiqi Li ◽  
John D. O’Brien
Keyword(s):  
Plasmodium Falciparum ◽  
Genetic Epidemiology ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Infected Individual ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Extensive Literature ◽  
Data Set ◽  
Inbreeding Coefficients

AbstractThe advent of whole-genome sequencing has generated increased interest in modeling the structure of strain mixture within clinicial infections of Plasmodium falciparum (Pf). The life cycle of the parasite implies that the mixture of multiple strains within an infected individual is related to the out-crossing rate across populations, making methods for measuring this process in situ central to understanding the genetic epidemiology of the disease. In this paper, we show how to estimate inbreeding coefficients using genomic data from Pf clinical samples, providing a simple metric for assessing within-sample mixture that connects to an extensive literature in population genetics and conservation ecology. Features of the P. falciparum genome mean that some standard methods for inbreeding coefficients and related F-statistics cannot be used directly. Here, we review an initial effort to estimate the inbreeding coefficient within clinical isolates of P. falciparum and provide several generalizations using both frequentist and Bayesian approaches. The Bayesian approach connects these estimates to the Balding-Nichols model, a mainstay within genetic epidemiology. We provide simulation results on the performance of the estimators and show their use on ~ 1500 samples from the PF3K data set. We also compare the results to output from a recent mixture model for within-sample strain mixture, showing that inbreeding coefficients provide a strong proxy for the results of these more complex models. We provide the methods described within an open-source R package pfmix.

scholarly journals 1615. Isolation of Lytic Bacteriophages with Broad Host Range Activity Against Pseudomonas aeruginosa Strains Isolated from Respiratory Samples from Cystic Fibrosis Patients Intended for Therapeutic Application

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S801-S801
Author(s):  
Jose Alexander ◽  
Daniel Navas ◽  
Marly Flowers ◽  
Angela Charles ◽  
Amy Carr
Keyword(s):  
Cystic Fibrosis ◽  
Host Range ◽  
Phage Therapy ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Plaque Morphology ◽  
Broad Host Range ◽  
Chronic Infections ◽  
Clinically Significant ◽  
Host Target

Abstract Background With the rise of the antimicrobial resistance between different genera and species of bacteria, Phage Therapy is becoming a more realistic and accessible option for patients with limited or no antimicrobial options. Being able to have rapid access to a collection of clinical active phages is key for rapid implementation of phage therapy. The Microbiology Department at AdventHealth Orlando is performing routine screening of environmental and patient samples for isolation of phages against non-fermenting Gram negative bacteria to develop a Phage Bank. Methods Protocols for phage isolation from environmental sources such as lakes, rivers and sewers and clinical samples were developed. A series of respiratory, throat, stool and urine samples were processed following an internal protocol that includes centrifugation, filtration and enrichment. Clinical samples were centrifugated for 10 minutes, filtered using 0.45µm centrifugation filters, seeded with targeted host bacteria (clinical isolates) and incubated at 35°C for 24 hours. The enriched samples were centrifugated and filtered for a final phage enriched solution. Screening and isolation were performed using the Gracia method over trypticase soybean agar (TSA) for plaque morphology and quantification. Host range screening of other clinical isolates of P. aeruginosa was performed using the new isolated and purified phages. Results 4 lytic phages against clinical strains of P. aeruginosa from patient with diagnosis of cystic fibrosis (CF), were isolated and purified from 4 different respiratory samples, including sputum and bronchial alveolar lavage. All phages showed phenotypical characteristics of lytic activity. 1 phage was active against 4 strains of P. aeruginosa, 1 phage was active against 2 strains of P. aeruginosa and the remaining 2 phages were active only against the initial host target strain. Conclusion With this study we demonstrated the potential use of clinical samples as source for isolating active bacteriophages against clinically significant bacteria strains. Clinical samples from vulnerable population of patients with chronic infections are part of our routine “phage-hunting” process to stock and grow our Phage Bank project for future clinical use. Disclosures All Authors: No reported disclosures

scholarly journals 1280. In-Vitro Activity of Cefiderocol, Imipenem/Relebactam, & Ceftazidime/Avibactam in Ceftolozane/Tazobactam Resistant Strains of Multidrug Resistant Pseudomonas aeruginosa

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S655-S655
Author(s):  
Daniel Navas ◽  
Angela Charles ◽  
Amy Carr ◽  
Jose Alexander
Keyword(s):  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Vitro Activity ◽  
Clinical Isolates ◽  
Resistance Testing ◽  
Clinical Samples ◽  
In Vitro Activity ◽  
Carbapenemase Gene ◽  
Resistant Strains

Abstract Background The activity of imipenem/relebactam (I/R), ceftazidime/avibactam (CZA) and cefiderocol (FDC) were evaluated against clinical isolates of multidrug resistant (MDR) strains of P. aeruginosa which was resistant to ceftolozane/tazobactam (C/T). The recent increase of MDR P. aeruginosa strains isolated from clinical samples has prompted research and development of new antimicrobials that can withstand its multiple resistance mechanisms. C/T is an effective option for treatment of MDR P. aeruginosa in our facility with only 10% of resistance in MDR strains, but the emergence of resistance may occur due to the presence of a carbapenemase gene or an ampC mutation. Methods Antimicrobial susceptibility testing for C/T Etest® (bioMérieux, Inc.) were performed on all MDR strains initially screened by the VITEK2® (bioMérieux, Inc.). 10% (n=20) of all MDR isolates were resistant to C/T by the CLSI 2019 breakpoints. These resistant isolates were tested for presence of a carbapenemase gene using the GeneXpert CARBA-R (Cepheid®) PCR and against CZA Etest® (bioMérieux, Inc.) I/R gradient strips (Liofilchem®) and FDC broth microdilution (Thermo Scientific™ Sensititre™). Results A total of 20 clinical isolates of MDR P. aeruginosa resistant to C/T were tested following standardized CLSI protocols and techniques. All 20 isolates were screened for the presence of a carbapenemase gene (blaVIM, blaNDM, blaKPC, blaOXA-48, blaIMP). A blaVIM gene was detected in 6 (30%) out of 20 isolates. FDC demonstrated the greatest activity with 85% (n=17) of susceptible isolates (CLSI MIC <4µg/dL). CZA (CLSI MIC <8µg/dL) and I/R (FDA MIC <2µg/dL) showed 15% (n=3) and 10% (n=2) of susceptible isolates respectively. FDC was active against all 6 blaVIM isolates, where all 6 strains were resistant to CZA and I/R as expected. 3 isolates tested non-susceptible against FDC; additional characterization was not performed at this time. Conclusion Based on these results, FDC demonstrated the greatest in-vitro activity against C/T resistant strains of MDR P. aeruginosa. FDC also demonstrated activity against all 6 MDR P. aeruginosa carrying blaVIM gene. FDC is a strong option to consider on MDR P. aeruginosa strains based on a resistance testing algorithm and a cost/effective protocol. Disclosures All Authors: No reported disclosures

scholarly journals Strain Typing of Trichosporon asahii Clinical Isolates by Random Amplification of Polymorphic DNA (RAPD) Analysis

2021 ◽  
Author(s):  
Thayanidhi Premamalini ◽  
Vijayaraman Rajyoganandh ◽  
Ramaraj Vijayakumar ◽  
Hemanth Veena ◽  
Anupma Jyoti Kindo ◽  
...  
Keyword(s):  
Genetic Relatedness ◽  
Rapd Analysis ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Strain Typing ◽  
Trichosporon Asahii ◽  
Pcr Fingerprinting ◽  
Specific Pcr ◽  
Rapd Primer ◽  
Random Amplification

Abstract Objective The aim of this study was to identify and isolate Trichosporon asahii (T. asahii) from clinical samples and to assess the genetic relatedness of the most frequently isolated strains of T. asahii using random amplification of polymorphic DNA (RAPD) primers GAC-1 and M13. Methods All the clinical samples that grew Trichosporon species, identified and confirmed by polymerase chain reaction (PCR) using Trichosporon genus-specific primers, were considered for the study. Confirmation of the species T. asahii was carried out by T. asahii-specific PCR. Fingerprinting of the most frequently isolated T. asahii isolates was carried out by RAPD using random primers GAC-1 and M13. Results Among the 72 clinical isolates of Trichosporon sp. confirmed by Trichosporon-specific PCR, 65 were found to be T. asahii as identified by T. asahii-specific PCR. Fingerprinting of the 65 isolates confirmed as T. asahii using GAC-1 RAPD primer yielded 11 different patterns, whereas that of M13 primer produced only 5 patterns. The pattern I was found to be the most predominant type (29.2%) followed by pattern III (16.9%) by GAC-1 primer. Conclusions This study being the first of its kind in India on strain typing of T. asahii isolates by adopting RAPD analysis throws light on genetic diversity among the T. asahii isolates from clinical samples. Fingerprinting by RAPD primer GAC-1 identified more heterogeneity among the T. asahii isolates than M13.

scholarly journals Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0121303 ◽  
Author(s):  
Miguel M. Pinheiro ◽  
Md Atique Ahmed ◽  
Scott B. Millar ◽  
Theo Sanderson ◽  
Thomas D. Otto ◽  
...  
Keyword(s):  
Plasmodium Knowlesi ◽  
Clinical Isolates ◽  
Genome Sequences

scholarly journals Within-Arctic horizontal gene transfer as a driver of convergent evolution in distantly related microalgae

2021 ◽  
Author(s):  
Richard G Dorrell ◽  
Alan Kuo ◽  
Zoltan Fussy ◽  
Elisabeth H Richardson ◽  
Asaf Salamov ◽  
...  
Keyword(s):  
Sequence Data ◽  
Sequence Similarity ◽  
Algal Species ◽  
Gene Families ◽  
The Arctic ◽  
Arctic Water ◽  
Diverse Range ◽  
Binding Domains ◽  
Ice Binding ◽  
Ice Conditions

The Arctic Ocean is being impacted by warming temperatures, increasing freshwater and highly variable ice conditions. The microalgal communities underpinning Arctic marine food webs, once thought to be dominated by diatoms, include a phylogenetically diverse range of small algal species, whose biology remains poorly understood. Here, we present genome sequences of a cryptomonad, a haptophyte, a chrysophyte, and a pelagophyte, isolated from the Arctic water column and ice. Comparing protein family distributions and sequence similarity across a densely-sampled set of algal genomes and transcriptomes, we note striking convergences in the biology of distantly related small Arctic algae, compared to non-Arctic relatives; although this convergence is largely exclusive of Arctic diatoms. Using high-throughput phylogenetic approaches, incorporating environmental sequence data from Tara Oceans, we demonstrate that this convergence was partly explained by horizontal gene transfers (HGT) between Arctic species, in over at least 30 other discrete gene families, and most notably in ice-binding domains (IBD). These Arctic-specific genes have been repeatedly transferred between Arctic algae, and are independent of equivalent HGTs in the Antarctic Southern Ocean. Our data provide insights into the specialised Arctic marine microbiome, and underlines the role of geographically-limited HGT as a driver of environmental adaptation in eukaryotic algae.

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