Metagenome Techniques for Detection of Pathogens Causing Ocular Infection

Metagenomic analysis is the comprehensive study of DNA using clinical specimens of organisms including bacteria, fungi, and viruses. In this study, we investigated the efficacy of metagenomic analysis for diagnosing ocular infections, including 11 keratitis cases, four iridocyclitis cases, and one endophthalmitis case. Corneal scraping, aqueous humor, and vitreous humor, were collected respectively. Ocular specimens were used for bacterial and fungal culture, and PCR for detecting viral DNA. Shotgun metagenomic sequencing for 150 bases of single end was performed by Illumina MiSeq® System. Sequence was retrieved from the database at NCBI using a MegaBLAST search. Since Propionibacterium spp. are commensal bacteria found at the ocular surface, they were excluded from analysis. Six cases (37.5%) were positive for culture or PCR. Metagenome techniques revealed that 9 cases (56.3%) included genomes of organisms that were considered pathogenic in specimens. Five cases (31.3%) possessed genomes of organisms like themselves that were detected by culture and PCR. Six cases (37.5%) were negative for culture, PCR, and metagenome analysis. Moreover, viral pathogens (HSV-1, 2 cases; and VZV, 1 case) were detected by only metagenome analysis. Metagenome analysis using an ocular sample can detect microbial genome comprehensively, and viral pathogens, which were not detected by conventional examination.

Download Full-text

Metagenome Analysis of Surface Waters of the Shardara Reservoir, the Largest Artificial Reservoir in Southern Kazakhstan

Microbiology Resource Announcements ◽

10.1128/mra.00053-20 ◽

2020 ◽

Vol 9 (11) ◽

Author(s):

Madina S. Alexyuk ◽

Andrey P. Bogoyavlenskiy ◽

Pavel G. Alexyuk ◽

Yergali S. Moldakhanov ◽

Vladimir E. Berezin

Keyword(s):

Microbial Community ◽

Surface Waters ◽

Metagenomic Analysis ◽

Metagenomic Sequencing ◽

Metagenome Analysis ◽

Artificial Reservoir ◽

Shotgun Metagenomic Sequencing ◽

Hydropower Engineering

Here, we present a metagenomic analysis of the microflora of the surface waters of the Shardara reservoir, the largest artificial reservoir in Southern Kazakhstan, created to meet irrigation and hydropower engineering needs. In this case, shotgun metagenomic sequencing of the microbial community DNA was used.

Download Full-text

Metagenomic Exploration of Koumiss from Kazakhstan

Microbiology Resource Announcements ◽

10.1128/mra.01082-21 ◽

2022 ◽

Author(s):

Andrey Bogoyavlenskiy ◽

Madina Alexyuk ◽

Pavel Alexyuk ◽

Makhabbat Amanbayeva ◽

Elmira Anarkulova ◽

...

Keyword(s):

Metagenomic Analysis ◽

Metagenomic Sequencing ◽

Viral Community ◽

Shotgun Metagenomic Sequencing ◽

Rna And Dna

Here, we report a metagenomic analysis of koumiss from Kazakhstan. In this study, shotgun metagenomic sequencing of the RNA and DNA viral community was performed.

Download Full-text

Metagenomic Exploration of Atelerix albiventris Gut Microbiome

Microbiology Resource Announcements ◽

10.1128/mra.01342-20 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Makhabbat Amanbayeva ◽

Elmira Anarkulova ◽

Andrey Bogoyavlenskiy ◽

Madina Alexyuk ◽

Anar Imangazy ◽

...

Keyword(s):

Gut Microbiome ◽

Metagenomic Analysis ◽

Metagenomic Sequencing ◽

Viral Community ◽

Shotgun Metagenomic Sequencing ◽

Rna And Dna

ABSTRACT Here, we report the metagenomic analysis of the gut of Atelerix albiventris, an animal typically kept as a pet in Kazakhstan. In this case, shotgun metagenomic sequencing of the RNA and DNA viral community was performed.

Download Full-text

Dual stain kit for the microscopic gram classification of ocular infection bacteria

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147089 ◽

1993 ◽

Vol 51 ◽

pp. 248-249

Author(s):

Jacob S. Hanker ◽

Dale N. Holdren ◽

Kenneth L. Cohen ◽

Beverly L. Giammara

Keyword(s):

Contact Lenses ◽

Ocular Infection ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Gram Staining ◽

Ocular Infections ◽

Different Types ◽

Culture Positive ◽

Increased Susceptibility

Keratitis and conjunctivitis (infections of the cornea or conjunctiva) are ocular infections caused by various bacteria, fungi, viruses or parasites; bacteria, however, are usually prominent. Systemic conditions such as alcoholism, diabetes, debilitating disease, AIDS and immunosuppressive therapy can lead to increased susceptibility but trauma and contact lens use are very important factors. Gram-negative bacteria are most frequently cultured in these situations and Pseudomonas aeruginosa is most usually isolated from culture-positive ulcers of patients using contact lenses. Smears for staining can be obtained with a special swab or spatula and Gram staining frequently guides choice of a therapeutic rinse prior to the report of the culture results upon which specific antibiotic therapy is based. In some cases staining of the direct smear may be diagnostic in situations where the culture will not grow. In these cases different types of stains occasionally assist in guiding therapy.

Download Full-text

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

Microorganisms ◽

10.3390/microorganisms9040707 ◽

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.

Download Full-text

Discriminating between JCPyV and BKPyV in Urinary Virome Data Sets

Viruses ◽

10.3390/v13061041 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1041

Author(s):

Rita Mormando ◽

Alan J. Wolfe ◽

Catherine Putonti

Keyword(s):

Jc Virus ◽

Sequence Similarity ◽

Bk Virus ◽

Data Sets ◽

Metagenomic Sequencing ◽

Significant Sequence Similarity ◽

Sequence Identity ◽

Shotgun Metagenomic Sequencing ◽

Urinary Microbiome ◽

Six Genes

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

Download Full-text

Fungal Keratitis in Northern Thailand: Spectrum of Agents, Risk Factors and Putative Virulence Factors

Journal of Fungi ◽

10.3390/jof7060475 ◽

2021 ◽

Vol 7 (6) ◽

pp. 475

Author(s):

Siriporn Chongkae ◽

Sirida Youngchim ◽

Joshua D. Nosanchuk ◽

Angkana Laliam ◽

Chulaluck Tangmonkongvoragul ◽

...

Keyword(s):

Virulence Factors ◽

Vision Loss ◽

Proteolytic Enzymes ◽

Tertiary Care ◽

Fungal Keratitis ◽

High Rate ◽

Ocular Infection ◽

Fungal Culture ◽

Care Hospital ◽

Northern Thailand

Fungal keratitis (FK) is a serious ocular infection that can result in various degrees of vision loss, including blindness. The aim of the study was to identify and retrospectively review all FK cases diagnosed between August 2012 and December 2020 at a tertiary care hospital in northern Thailand with a specific focus on epidemiologic features, including season, patient sex and age, the spectrum of pathogens, and presence of certain putative virulence factors. Of 1237 patients with corneal ulcers, 294 (23.8%) were confirmed by direct microscopic examination and/or fungal culture. For the positive cases, direct examinations of Calcofluor white (CW) stains and KOH mounts were found in 97.3% (286/294) and 76.5% (225/294), respectively (p < 0.05). Of the cases diagnosed by microscopy and culture, fungi were isolated in 152 (51.7%), with Fusarium spp. being the most frequently identified (n = 69, 45.5%) followed by dematiaceous fungi (n = 45, 29.6%) and Aspergillus spp. (n = 18, 11.8%). The incidence of FK was higher in the rainy season of July to October. The mean age was 54.4 ± 14.4 (SD) years, with a range of 9–88 years. Males (75.8%) were affected significantly more than females (24.2%) (p < 0.05). Of 294 patients, 132 (44.9%) were middle-aged adults (41–60 years) and 107 (36.4%) were older than 60 years. Trauma to the eye by soil or vegetative matter were the most common preceding factors (188/294; 64.0%). We assessed two virulence factors. First, 142 of the 152 culture-positive FK cases were due to molds, indicating that hyphal morphogenesis is extremely important in disease. We also demonstrated that fungal melanization occurs in the molds during the course of FK by applying a melanin-specific monoclonal antibody (MAb) that labeled fungal elements in corneal samples of patients, and melanin particles derived from the hyphae were also recovered after treatment of the samples with proteolytic enzymes, denaturant and hot concentrated acid. In summary, we demonstrate that northern Thailand has a high rate of FK that is influenced by season and males engaged in outside activities are at highest risk for disease. Moulds are significantly more commonly responsible for FK, in part due to their capacity to form hyphae and melanins. Future studies will examine models of fungal corneal interactions and assess additional factors of virulence, such as secreted enzymes, to more deeply decipher the pathogenesis of FK.

Download Full-text

DNA extraction from concrete v2

10.17504/protocols.io.b2i5qcg6 ◽

2021 ◽

Author(s):

Anders Kiledal ◽

Julia A Maresca

Keyword(s):

Dna Extraction ◽

Dental Plaque ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Metagenomic Sequencing ◽

Sample Analysis ◽

Larger Sample ◽

Laboratory Contamination ◽

Biomass Sample ◽

Over Time

This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

Download Full-text

Evaluation of the CosmosID Bioinformatics Platform for Prosthetic Joint-Associated Sonicate Fluid Shotgun Metagenomic Data Analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.01182-18 ◽

2018 ◽

Vol 57 (2) ◽

Cited By ~ 8

Author(s):

Qun Yan ◽

Yu Mi Wi ◽

Matthew J. Thoendel ◽

Yash S. Raval ◽

Kerryl E. Greenwood-Quaintance ◽

...

Keyword(s):

Antibiotic Resistance ◽

Metagenomic Data ◽

Metagenomic Sequencing ◽

Antibacterial Resistance ◽

Sequencing Data ◽

Bacterial Detection ◽

Shotgun Metagenomic Sequencing ◽

Prosthetic Joint ◽

Validation Set ◽

Fluid Culture

ABSTRACT We previously demonstrated that shotgun metagenomic sequencing can detect bacteria in sonicate fluid, providing a diagnosis of prosthetic joint infection (PJI). A limitation of the approach that we used is that data analysis was time-consuming and specialized bioinformatics expertise was required, both of which are barriers to routine clinical use. Fortunately, automated commercial analytic platforms that can interpret shotgun metagenomic data are emerging. In this study, we evaluated the CosmosID bioinformatics platform using shotgun metagenomic sequencing data derived from 408 sonicate fluid samples from our prior study with the goal of evaluating the platform vis-à-vis bacterial detection and antibiotic resistance gene detection for predicting staphylococcal antibacterial susceptibility. Samples were divided into a derivation set and a validation set, each consisting of 204 samples; results from the derivation set were used to establish cutoffs, which were then tested in the validation set for identifying pathogens and predicting staphylococcal antibacterial resistance. Metagenomic analysis detected bacteria in 94.8% (109/115) of sonicate fluid culture-positive PJIs and 37.8% (37/98) of sonicate fluid culture-negative PJIs. Metagenomic analysis showed sensitivities ranging from 65.7 to 85.0% for predicting staphylococcal antibacterial resistance. In conclusion, the CosmosID platform has the potential to provide fast, reliable bacterial detection and identification from metagenomic shotgun sequencing data derived from sonicate fluid for the diagnosis of PJI. Strategies for metagenomic detection of antibiotic resistance genes for predicting staphylococcal antibacterial resistance need further development.

Download Full-text

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples

10.1101/2021.05.04.21256618 ◽

2021 ◽

Author(s):

Jutte J.C. de Vries ◽

Julianne R. Brown ◽

Nicole Fischer ◽

Igor A. Sidorov ◽

Sofia Morfopoulou ◽

...

Keyword(s):

De Novo ◽

Respiratory Infections ◽

Limit Of Detection ◽

Bioinformatic Analysis ◽

Clinical Samples ◽

Mixed Infections ◽

Metagenomic Sequencing ◽

User Friendliness ◽

Viral Pathogens ◽

Clinical Diagnostic

Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Methods Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analysed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analysed. Results Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. Conclusion A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

Download Full-text