Finding Sarcocystis spp. on the Tioman Island: 28S rRNA gene next-generation sequencing reveals nine new Sarcocystis species

Abstract The Tioman Island of Malaysia experienced acute muscular sarcocystosis outbreaks from 2011 to 2014. So far, a previous study based on the 18S rRNA gene sequencing has reported S. singaporensis, S. nesbitti and Sarcocystis sp. YLL-2013 in water samples acquired from the island, thus confirming the waterborne nature of this emerging parasitic disease. This study aimed to improve the detection methods for Sarcocystis, in order to have a clearer picture of the true diversity of Sarcocystis species in Tioman. A new primer set (28S R7F–28S R8 Deg R) was designed to amplify the 28S rRNA gene of Sarcocystis. Subsequently, Sarcocystidae was detected in 65.6% (21/32) of water samples and 28% (7/25) of soil samples acquired between 2014 and 2015 from Tioman. Next-generation sequencing (NGS) on 18 of the positive samples was then performed using amplicons generated from the same primer set. This yielded 53 potentially unique Sarcocystidae sequences (290 bp), of which nine of the most abundant, prevalent and unique sequences were named herein. In contrast, NGS of the 18S rRNA gene V9 hypervariable region of 10 selected samples detected only two Sarcocystis species (160 bp). S. mantioni was the most ubiquitous sequence found in this study.

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Bacterial abundance and community composition in green, brown and red water from intensive catfish (Clarias sp.) culture ponds in Yogyakarta, Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d220910 ◽

2021 ◽

Vol 22 (9) ◽

Author(s):

Karunia Adetera Nungki Wijayanti ◽

Indah Istiqomah ◽

Murwantoko Murwantoko

Keyword(s):

Next Generation Sequencing ◽

Bacterial Community ◽

16S Rrna ◽

Community Composition ◽

Water Samples ◽

Bacterial Abundance ◽

Rrna Gene ◽

Next Generation ◽

Aquaculture System ◽

Generation Sequencing

Abstract. Wijayanti KAN, Istiqomah I, Murwantoko. 2021. Bacterial abundance and community composition in green, brown and red water from intensive Catfish (Clarias sp.) culture ponds in Yogyakarta, Indonesia. Biodiversitas 22: 3677-3684. Catfish (Clarias sp.) is an important aquaculture commodity in Indonesia and cultured in an intensive system. Microorganisms play an important role in maintaining water quality of aquaculture system. The objective of this study was to determine the bacterial abundance and community composition of green, brown and red water collected from intensive catfish culture ponds in Yogyakarta using next-generation sequencing method. The water samples were collected from intensive catfish culture ponds with different colors, namely green, brown and red ponds located in Yogyakarta. The DNA from water samples was extracted using DNA extraction kit and used as template for 16S rRNA amplification. The V3-V4 hypervariable regions of the 16S rRNA gene were amplified apply for next-generation sequencing technology. This study could explore effectively the bacterial community in water samples. The bacterial communities in this catfish culture water showed higher bacterial richness compared to the other aquaculture system. The diversity of the green, brown and red catfish culture water ponds was similar with the number OTUs of the green, brown and red water samples, which were 1269; 1387 and 1323 OTUs respectively. The 694 OTUs (34.42%) were common core microbiomes in all catfish culture ponds, the 212 OTUs (10.51%) are present on green and brown water ponds, the 182 OTUs (9.02%) were on green and red water ponds, and the 183 OTUs (9.07%) were present on green and brown water ponds. However, the composition of the bacterial community was different. The most dominant phylum in green and brown water ponds was Proteobacteria with relative abundance in green water and brown water 71.6% and 47.0% respectively, whereas, the most dominant phylum in red water was Firmicutes (29.5%). The dominance of Firmicutes phylum in red water ponds may be caused by application of probiotic bacteria, the high organic content, and low oxygen concentration.

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Sequencing and Analysis of Fungal rRNA Operons for Development of Broad-Range Fungal PCR Assays

Applied and Environmental Microbiology ◽

10.1128/aem.02383-08 ◽

2009 ◽

Vol 75 (6) ◽

pp. 1559-1565 ◽

Cited By ~ 52

Author(s):

Prasanna D. Khot ◽

Daisy L. Ko ◽

David N. Fredricks

Keyword(s):

Fungal Pathogens ◽

18S Rrna ◽

18S Rrna Gene ◽

Internal Transcribed Spacers ◽

Rrna Gene ◽

28S Rrna ◽

28S Rrna Gene ◽

Fungal Dna ◽

Pcr Assays ◽

Distance Matrices

ABSTRACT rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5′ end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S rRNA gene remains largely unexplored because sequences of many medically relevant fungi are either unavailable or undefined in genomic databases. The internal transcribed spacers and 28S rRNA gene of nine medically and phylogenetically important fungi were sequenced. In addition, 42 sequences from this region were acquired from public databases, resulting in an alignment of 51 fungal sequences spanning 30 fungal genera. For the nearly 3,950-bp region from the 3′ end of 18S rRNA gene to the 3′ end of the 28S rRNA gene, 27 broad-range PCR primers were designed such that their sequence homology with the human rRNA gene was minimal. All 62 possible amplicons in the size range from 75 to 400 bp from 27 primers were screened using fungal genomic DNA from 26 species spanning 14 genera. Eleven of the 62 amplicons did not cross-react with 1 μg/PCR human DNA but simultaneously amplified 10 fg of fungal DNA. Phylogenetic distance matrices were calculated for regions covered by these 11 amplicons based on 51 fungi. Two of these 11 amplicons successfully amplified 30 fg of fungal DNA from 25 of 26 fungi and provided the most phylogenetic information for species identification based on the distance matrices. These PCR assays hold promise for detection and identification of fungal pathogens in human tissues.

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Diversity of cultured photosynthetic flagellates in the North East Pacific and Arctic Oceans in summer

Biogeosciences Discussions ◽

10.5194/bgd-9-6219-2012 ◽

2012 ◽

Vol 9 (6) ◽

pp. 6219-6259 ◽

Cited By ~ 3

Author(s):

S. Balzano ◽

P. Gourvil ◽

R. Siano ◽

M. Chanoine ◽

D. Marie ◽

...

Keyword(s):

18S Rrna ◽

Gene Sequencing ◽

18S Rrna Gene ◽

Rrna Gene ◽

28S Rrna ◽

28S Rrna Gene ◽

North East ◽

The North ◽

Green Algal ◽

Rrna Gene Sequencing

Abstract. During the MALINA cruise (summer 2009) an extensive effort was undertaken to isolate phytoplankton strains from the North East (NE) Pacific Ocean, the Bering Strait, and the Beaufort Sea. Strains were isolated by flow cytometry sorting (FCS) and pipetting before or after phytoplankton enrichment of seawater samples. Strains were isolated both onboard and back in the laboratory and cultured at 4 °C under light/dark conditions. Overall, we isolated and characterised by light microscopy and 18S rRNA gene sequencing 104 strains of photosynthetic flagellates which grouped into 21 genotypes (defined by 99.5% 18S rRNA gene sequence similarity) mainly affiliated to Chlorophyta and Heterokontophyta. The taxon most frequently isolated was an Arctic ecotype of the green algal genus Micromonas (Arctic Micromonas) which was almost the only phytoplankter recovered within picoplankton (≤ 2 μm) size range. Strains of Arctic Micromonas as well as three unidentified strains related to the same genus were identified in further details by sequencing the Internal Transcribed Spacer (ITS) region of the rRNA operon. The MALINA Micromonas strains share identical 18S rRNA and ITS sequences suggesting high genetic homogeneity within Arctic Micromonas. The unidentified strains form a genotype likely belonging to a new genus within the family Mamiellaceae to which Micromonas belongs. Other green algae genotypes from the genera Nephroselmis, Chlamydomonas, Pyramimonas were also isolated whereas Heterokontophyta included Pelagophyceae, Dictyochophyceae and Chrysophyceae. Dictyochophyceae included Pedinellales which could not be identified to the genus level whereas Chrysophyceae comprised Dinobryon faculiferum. Moreover, we isolated Rhodomonas sp. as well as a few Haptophyta and dinoflagellates. We identified the dinoflagellate Woloszynskia cincta by Scanning Electron Microscopy (SEM) and 28S rRNA gene sequencing. Our morphological analyses show that this species possess the diagnostic features of the genus Biecheleria, and the 28S rRNA gene topology corroborates this affiliation. We thus propose the transfer of W. cincta to the genus Biecheleria and its recombination as Biecheleria cincta.

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Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

BMC Veterinary Research ◽

10.1186/s12917-021-02969-9 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Elton J. R. Vasconcelos ◽

Chayan Roy ◽

Joseph A. Geiger ◽

Kristina M. Oney ◽

Melody Koo ◽

...

Keyword(s):

Veterinary Medicine ◽

Next Generation Sequencing ◽

Complete Analysis ◽

Molecular Diagnostic ◽

Rrna Gene ◽

Spotted Fever Group ◽

Next Generation ◽

Vector Borne ◽

16 S Rrna ◽

Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

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Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

Microorganisms ◽

10.3390/microorganisms9010021 ◽

2020 ◽

Vol 9 (1) ◽

pp. 21

Author(s):

Abdul Ghafar ◽

Anson V. Koehler ◽

Ross S. Hall ◽

Charles G. Gauci ◽

Robin B. Gasser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Water Buffalo ◽

Hypervariable Region ◽

Small Subunit ◽

Rrna Gene ◽

Next Generation ◽

Small Subunit Rrna ◽

Tropical Theileriosis ◽

Ecological Zones ◽

Generation Sequencing

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

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Next-Generation Sequencing for Pathogen Identification in Infected Foot Ulcers

Foot & Ankle Orthopaedics ◽

10.1177/2473011420s00027 ◽

2020 ◽

Vol 5 (4) ◽

pp. 2473011420S0002

Author(s):

Yoonjung Choi ◽

Irvin Oh

Keyword(s):

Next Generation Sequencing ◽

False Positive ◽

Bacterial Genome ◽

Rrna Gene ◽

Next Generation ◽

Antibiotic Resistant ◽

Streptococcus Species ◽

Resistant Genes ◽

Standard Culture ◽

Generation Sequencing

Category: Other Introduction/Purpose: Foot infections are often polymicrobial with diverse microbiomes. Accurate identification of the main pathogen in diabetic foot ulcer (DFU) remain challenging due to contamination or negative cultures often leading to ineffective post-surgical antibiotic treatment. Application of molecular diagnostics, such as next generation sequencing (NGS) has been explored as an alternative to standard culture in orthopaedic infections. NGS is highly sensitive and detects an entire bacterial genome along with pharmacologic resistant genes in a given sample. We sought to investigate the potential use of NGS for accurate diagnosis and quantification of various species in infected DFU. We hypothesize that NGS will provide a more accurate means of diagnosing and profiling microorganisms in infected DFU compared to the standard culture method. Methods: We investigated 30 infected DFU patients who underwent surgical treatment by a single academic orthopaedic surgeon from October 2018 to September 2019. The average age of the patient was 60.4 (range 33-82) years-old. Surgical procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Quantitative PCR was performed to determine the bacterial burden present in the sample. DNA was amplified by PCR from a highly conserved region of the rRNA gene in the bacteria (16S rRNA). Once a high level of DNA was generated and determined, it was compared against NIH GenBank database. Concordance between the standard culture and NGS was assessed. Results: In 28 of 29 patients, pathogens were identified by both NGS and culture, with complete consistency of organisms in 13 cases (concordance rate: 43.3%). NGS provided relative quantitative measures and the presence of antibiotic resistant genes for each pathogen. In NGS, Anaerococcus species (79.3%) was the most common organism, followed by Streptococcus species (44.8%), Prevotella species (44.8%), Finegoldia magna (44.8%). In culture, S. aureus (58.6%) was the most common, followed by Streptococcus species (34.5%), coagulase-negative Staphylococci (24.1%), Corynebacterium species (20.7%). On average, NGS revealed 5.1 (1-11) number of pathogens, whereas standard culture revealed 2.6 (1-6) pathogens in a given sample. NGS identified 2 cases with false positive standard culture and detected antibiotic resistant organisms in 15 specimens. Conclusion: NGS is an emerging method of microbial identification in orthopedic infection. It is particularly helpful in profiling diverse microbes in polymicrobial infected DFU. It can identify major pathogens and may correct false positive or false negative culture. NGS may allow a faster invitation of postoperative targeted antibiotic therapy. [Table: see text]

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Serra da Estrela PDO Cheese Microbiome as Revealed by Next Generation Sequencing

Microorganisms ◽

10.3390/microorganisms9102007 ◽

2021 ◽

Vol 9 (10) ◽

pp. 2007

Author(s):

Rui Rocha ◽

Manuela Vaz Velho ◽

Joana Santos ◽

Paulo Fernandes

Keyword(s):

Next Generation Sequencing ◽

Relative Abundance ◽

Raw Materials ◽

Rrna Gene ◽

Next Generation ◽

Candida Spp ◽

Characterization Study ◽

Materials Used ◽

Serra Da Estrela ◽

Generation Sequencing

Serra da Estrela PDO cheese is the oldest traditional cheese manufactured in Portugal. In this work, its microbiome as well as the main raw materials used in cheese production, raw ewes’ milk and thistle flowers (Cynara cardunculus L.), were characterized using next generation sequencing. Samples were accordingly retrieved from a local producer over two consecutive production campaigns and at different time periods within each campaign. The bacterial and fungi communities associated with each matrix were accessed through sequencing of V3−V4 and Internal Transcribed Spacer 2 regions of rRNA gene amplicons, respectively. A high microbial diversity was found associated to each matrix, differing significantly (p < 0.05) from each other. Over 500 taxa were identified in each analyzed matrix, ranging from dominant (relative abundance > 1%), sub-dominant (0.01−1%) and rare taxa (<0.01%). Specifically, in cheese, 30 taxa were present in all analyzed samples (core taxa), including species of Leuconostoc spp. and Lactococcus spp. for bacteria and Candida spp., Debaryomyces spp. and Yarrowia spp. for fungi, that were cumulatively the most prevalent genera in Serra da Estrela PDO cheese (average relative abundance ≥10%). Ultimately, this characterization study may contribute to a better understanding of the microbial dynamics of this traditional PDO product, namely the influence of raw materials on cheese microbiome, and could assist producers interested in preserving the identity, quality and safety of Serra da Estrela PDO cheese.

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Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009779 ◽

2021 ◽

Vol 15 (10) ◽

pp. e0009779

Author(s):

Fakhriddin Sarzhanov ◽

Funda Dogruman-Al ◽

Monica Santin ◽

Jenny G. Maloney ◽

Ayse Semra Gureser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Gastrointestinal Symptoms ◽

Molecular Methods ◽

Clinical Samples ◽

Rrna Gene ◽

Next Generation ◽

Dientamoeba Fragilis ◽

Significant Difference ◽

Generation Sequencing ◽

Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

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Morphological and molecular characterization of Mermisnigrescens Dujardin, (Nematoda: Mermithidae) parasitizing the introduced European earwig (Dermaptera: Forficulidae) in New Zealand

Journal of Helminthology ◽

10.1017/s0022149x14000017 ◽

2014 ◽

Vol 89 (3) ◽

pp. 267-276 ◽

Cited By ~ 7

Author(s):

B. Presswell ◽

S. Evans ◽

R. Poulin ◽

F. Jorge

Keyword(s):

New Zealand ◽

18S Rrna ◽

Sequence Data ◽

18S Rrna Gene ◽

Parasitic Nematodes ◽

Rrna Gene ◽

28S Rrna ◽

Forficula Auricularia ◽

Adult Females ◽

European Earwig

AbstractParasitic nematodes of the family Mermithidae were found to be infecting the introduced European earwig Forficula auricularia (Dermaptera: Forficulidae) in Dunedin, South Island, New Zealand. Adult females were later collected from various garden plants while depositing eggs. These mermithid specimens were identified morphologically as Mermis nigrescens Dujardin, 1842. A genetic distance of 0.7% between these specimens and a M. nigrescens isolate from Canada (18S rRNA gene), suggests that they have diverged genetically, but there are currently no available comparable sequences for the European M. nigrescens. Two additional nuclear fragments were also amplified, the 28S rRNA and the ribosomal DNA first internal transcribed spacer (ITS1), providing a basis for future studies. Bearing in mind the morphological similarity with other reported M. nigrescens and the lack of sequence data from other parts of the world, we retain the name M.nigrescens, and suggest that the species may be found to represent a complex of cryptic species when more worldwide data are available. Herein, we present a brief description of the post-parasitic worms and adult females, along with an inferred phylogeny using 18S rRNA gene sequences.

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