scholarly journals Cryptococcus neoformans and Other Opportunistic Cryptococcus Species in Pigeon Dropping in Saudi Arabia: Identification and Characterization by DNA Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Bashir Sirag ◽  
El-Shiekh Khidir ◽  
Mohammed Dumyati ◽  
Basam Sindi ◽  
Mahir Alsinnari ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Cryptococcus Neoformans ◽  
Variant Calling ◽  
Illumina Miseq ◽  
Chromosome 1 ◽  
Susceptible Population ◽  
Genomic Epidemiology ◽  
Domestic Bird ◽  
Cryptococcus Species ◽  
Pigeon Dropping

The prevalent variants of Cryptococcus neoformans, and other Cryptococcus species in pigeon excreta in Western Region of Saudi Arabia were studied. Ninety pigeon dropping samples were plated directly on Niger seed agar, and suspected colonies were sequenced using Illumina MiSeq. Species identification was determined using sequence read mapping to reference genomes of the two C. neoformans variants. In addition, sequence reads were identified using the KmerFinder tool. internal transcribed spacer 2 in the rDNA was also used for fungal barcoding of none of the C. neoformans species using two fungal identification databases. Phylogeny was studied using CSI Phylogeny (Center for Genomic Epidemiology, Denmark). The C. neoformans var. grubii mitochondrion and chromosome 1 reference sequences (accession numbers NC_004336.1 and CP022321.1, respectively) were used for sequence comparison and variant calling. Fifteen Cryptococcus isolates were isolated, 11 were identified as C. neoformans var. grubii, and 4 were found to be other opportunistic Cryptococcus species. Phylogeny analysis of C. neoformans var. grubii isolates showed a high degree of similarity between the C. neoformans isolates especially at the mitochondrial genome level. This study supports the fact that pathogenic and opportunistic Cryptococcus species are prevalent in domestic bird excreta which is an easy source of infection in the susceptible population.

scholarly journals Characterization of Cryptococcus neoformans isolated from urban environmental sources in Goiânia, Goiás State, Brazil

2005 ◽  
Vol 47 (4) ◽  
pp. 203-207 ◽  
Author(s):  
Cláudia Castelo Branco Artiaga Kobayashi ◽  
Lúcia Kioko Hasimoto e Souza ◽  
Orionalda de Fátima Lisboa Fernandes ◽  
Sula Cristina Assis de Brito ◽  
Ana Cláudia Silva ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Cryptococcus Species ◽  
Major Route ◽  
Opportunistic Fungal Pathogen ◽  
Ornamental Trees ◽  
Pigeon Dropping ◽  
Vegetable Material

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis as the most frequent clinical presentation in immunocompromised patients, mainly in people infected by HIV. This fungus is an environmental encapsulated yeast, commonly found in soil enriched with avian droppings and plant material. A total of 290 samples of pigeon and the other avian droppings, soil, ornamental trees and vegetable material associated with Eucalyptus trees were collected to study environmental sources of Cryptococcus species in Goiânia, Goiás State. The determination of varieties, serotypes and the susceptibility in vitro to fluconazole, itraconazole and amphotericin B of C. neoformans isolates were performed. C. neoformans var. grubii (serotype A) was found in 20.3% (36/177) of pigeon dropping samples and in 14.3% (5/35) of samples of Eucalyptus. None of the environmental isolates of C. neoformans showed in vitro resistance to three antifungal agents. The knowledge of major route for human cryptococcal infection (inhalation of infectious particles from saprophytic sources) and a total of 60 C. neoformans isolates obtained from AIDS patients with cryptococcal meningitis between October 2001 and April 2002 justify the study of the habitats of these yeasts as probable sources of cryptococcosis in this city.

scholarly journals Low Glucose Mediated Fluconazole Tolerance in Cryptococcus neoformans

2021 ◽  
Vol 7 (6) ◽  
pp. 489
Author(s):  
Somanon Bhattacharya ◽  
Natalia Kronbauer Oliveira ◽  
Anne G. Savitt ◽  
Vanessa K. A. Silva ◽  
Rachel B. Krausert ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mitochondrial Function ◽  
Efflux Pump ◽  
Nile Red ◽  
Fold Increase ◽  
Growth Conditions ◽  
Chromosome 1 ◽  
Normal Glucose ◽  
Low Glucose ◽  
Synthetic Media

Chronic meningoencephalitis is caused by Cryptococcus neoformans and is treated in many parts of the world with fluconazole (FLC) monotherapy, which is associated with treatment failure and poor outcome. In the host, C. neoformans propagates predominantly under low glucose growth conditions. We investigated whether low glucose, mimicked by growing in synthetic media (SM) with 0.05% glucose (SMlowglu), affects FLC-resistance. A > 4-fold increase in FLC tolerance was observed in seven C. neoformans strains when minimum inhibitory concentration (MIC) was determined in SMlowglu compared to MIC in SM with normal (2%) glucose (SMnlglu). In SMlowglu, C. neoformans cells exhibited upregulation of efflux pump genes AFR1 (8.7-fold) and AFR2 (2.5-fold), as well as decreased accumulation (2.6-fold) of Nile Red, an efflux pump substrate. Elevated intracellular ATP levels (3.2-fold and 3.4-fold), as well as decreased mitochondrial reactive oxygen species levels (12.8-fold and 17-fold), were found in the presence and absence of FLC, indicating that low glucose altered mitochondrial function. Fluorescence microscopy revealed that mitochondria of C. neoformans grown in SMlowglu were fragmented, whereas normal glucose promoted a reticular network of mitochondria. Although mitochondrial membrane potential (MMP) was not markedly affected in SMlowglu, it significantly decreased in the presence of FLC (12.5-fold) in SMnlglu, but remained stable in SMlowglu-growing C. neoformans cells. Our data demonstrate that increased FLC tolerance in low glucose-growing C. neoformans is the result of increased efflux pump activities and altered mitochondrial function, which is more preserved in SMlowglu. This mechanism of resistance is different from FLC heteroresistance, which is associated with aneuploidy of chromosome 1 (Chr1).

scholarly journals Cryptococcus Species Other Than Cryptococcus neoformans and Cryptococcus gattii: Are They Clinically Significant?

2020 ◽  
Vol 7 (12) ◽  
Author(s):  
Edison J Cano ◽  
Zachary A Yetmar ◽  
Raymund R Razonable
Keyword(s):  
Cryptococcus Neoformans ◽  
Opportunistic Infections ◽  
Descriptive Analysis ◽  
Laboratory Data ◽  
Invasive Disease ◽  
Cryptococcus Gattii ◽  
Cryptococcus Laurentii ◽  
Cryptococcus Species ◽  
Clinically Significant ◽  
Cryptococcus Spp

Abstract Background Cryptococcus spp is a major cause of opportunistic infections in immunocompromised patients, primarily due to Cryptococcus neoformans and Cryptococcus gattii. There are occasional reports of other Cryptococcus species causing invasive human disease. However, their epidemiology and clinical significance are not fully defined. We sought to describe cases with cultures positive for Cryptococcus species other than C neoformans and C gattii. Methods A retrospective descriptive analysis of clinical and laboratory data of patients with cultures growing Cryptococcus species other than C neoformans and C gattii from November 2011 to February 2019 was performed. Three Mayo Clinic sites in Arizona, Florida, and Minnesota were included. Results From 176 cases with a culture growing Cryptococcus spp, 54 patients (30%) had a culture for Cryptococcus other than C neoformans and C gattii in the study time frame. The most common species were Cryptococcus magnus, Cryptococcus laurentii, and Cryptococcus ater. The organisms were isolated and identified in culture of bronchoalveolar lavage (11), skin (11), urine (7), oral (4), sinus (3), intraoperative soft tissue (3), sputum (2), synovial fluid (2), cerebrospinal fluid (2), and intravenous catheter (2), among others (7). Only 8 (15%) cases were considered to be potentially pathogenic, with 1 case of invasive disease. Antifungal treatment was fluconazole, itraconazole, and griseofulvin, for a mean systemic antifungal duration of 42 days. Conclusions This large series of patients with Cryptococcus spp other than C neoformans and C gattii suggests that these species rarely cause clinically significant infection in humans. Only 1 case of invasive disease was found.

scholarly journals Analyzing Low-Level mtDNA Heteroplasmy—Pitfalls and Challenges from Bench to Benchmarking

2021 ◽  
Vol 22 (2) ◽  
pp. 935
Author(s):  
Federica Fazzini ◽  
Liane Fendt ◽  
Sebastian Schönherr ◽  
Lukas Forer ◽  
Bernd Schöpf ◽  
...  
Keyword(s):  
Variant Calling ◽  
Illumina Miseq ◽  
Bioinformatic Analysis ◽  
Low Level ◽  
Sequencing Technologies ◽  
Laboratory Setup ◽  
Dna Mixture ◽  
The Individual ◽  
Highly Sensitive Detection ◽  
Sensitivity Specificity

Massive parallel sequencing technologies are promising a highly sensitive detection of low-level mutations, especially in mitochondrial DNA (mtDNA) studies. However, processes from DNA extraction and library construction to bioinformatic analysis include several varying tasks. Further, there is no validated recommendation for the comprehensive procedure. In this study, we examined potential pitfalls on the sequencing results based on two-person mtDNA mixtures. Therefore, we compared three DNA polymerases, six different variant callers in five mixtures between 50% and 0.5% variant allele frequencies generated with two different amplification protocols. In total, 48 samples were sequenced on Illumina MiSeq. Low-level variant calling at the 1% variant level and below was performed by comparing trimming and PCR duplicate removal as well as six different variant callers. The results indicate that sensitivity, specificity, and precision highly depend on the investigated polymerase but also vary based on the analysis tools. Our data highlight the advantage of prior standardization and validation of the individual laboratory setup with a DNA mixture model. Finally, we provide an artificial heteroplasmy benchmark dataset that can help improve somatic variant callers or pipelines, which may be of great interest for research related to cancer and aging.

scholarly journals PrimerPooler: automated primer pooling to prepare library for targeted sequencing

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Silas S. Brown ◽  
Yun-Wen Chen ◽  
Ming Wang ◽  
Alexandra Clipson ◽  
Eguzkine Ochoa ◽  
...  
Keyword(s):  
Variant Calling ◽  
Pcr Amplification ◽  
Illumina Miseq ◽  
Degenerate Primers ◽  
Cross Hybridization ◽  
Targeted Next Generation Sequencing ◽  
Target Sequencing ◽  
A Genome ◽  
Experimental Protocols ◽  
Minimal Depth

Abstract Targeted next-generation sequencing based on PCR amplification involves pooling of hundreds to thousands of primers, for preamplification and subsequent parallel single/multiplex PCR. It is often necessary to allocate the set of primers into subpools, a common issue being potential cross-hybridization. For smaller numbers of primers, pool division can be done manually using trial and error to minimize potential hybridization, but this becomes inefficient and time consuming with increasing numbers of primer pairs. We developed PrimerPooler that automates swapping of primer pairs between any user-defined number of subpools to obtain combinations with low-potential interactions. PrimerPooler performs inter-/intra-primer hybridization analysis to identify the adverse interactions, as well as simultaneous mapping of all primers onto a genome sequence in a single run without requiring a prior index of the genome. This allows detection of overlapping primer pairs and allocation of these primer pairs into separate subpools where tiling approaches are used. Using PrimerPooler, 1153 primer pairs were assigned to three preamplification pools (388, 389 and 376 primer pairs each), then 144 subpools of six- to nine-plex PCR for Fluidigm Access Array PCR, followed by Illumina MiSeq sequencing. With optimized experimental protocols, an average of 3269 reads was achieved for the targeted regions, with 95% of targets covered by at least 50 reads, the minimal depth of reads for confident variant calling. PrimerPooler provides a fast and highly efficient stratification of primer pairs for targeted enrichment, thus ensuring representative amplification of the targeted sequences. PrimerPooler is also able to analyse degenerate primers, and is thus also useful for microbiological identification and related target sequencing.

scholarly journals Genomic variant identification methods alter Mycobacterium tuberculosis transmission inference

2019 ◽  
Author(s):  
Katharine S. Walter ◽  
Caroline Colijn ◽  
Ted Cohen ◽  
Barun Mathema ◽  
Qingyun Liu ◽  
...  
Keyword(s):  
Sequence Data ◽  
Variant Calling ◽  
Genomic Variation ◽  
Human Pathogens ◽  
Phylogenetic Structure ◽  
Genomic Epidemiology ◽  
Tuberculosis Transmission ◽  
Epidemiology Studies ◽  
Global And Local ◽  
Variant Identification

AbstractPathogen genomic data are increasingly used to characterize global and local transmission patterns of important human pathogens and to inform public health interventions. Yet there is no current consensus on how to measure genomic variation. We investigated the effects of variant identification approaches on transmission inferences for M. tuberculosis by comparing variants identified by five different groups in the same sequence data from a clonal outbreak. We then measured the performance of commonly used variant calling approaches in recovering variation in a simulated tuberculosis outbreak and tested the effect of applying increasingly stringent filters on transmission inferences and phylogenies. We found that variant calling approaches used by different groups do not recover consistent sets of variants, often leading to conflicting transmission inferences. Further, performance in recovering true outbreak variation varied widely across approaches. Finally, stringent filters rapidly eroded the accuracy of transmission inferences and quality of phylogenies reconstructed from outbreak variation. We conclude that measurements of genetic distance and phylogenetic structure are dependent on variant calling approach. Variant calling algorithms trained upon true sequence data outperform other approaches and enable inclusion of repetitive regions typically excluded from genomic epidemiology studies, maximizing the information gleaned from outbreak genomes.

scholarly journals 377. SARS-CoV-2 Genomic Surveillance Reveals Little Spread Between a Large University Campus and the Surrounding Community

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S290-S290
Author(s):  
Andrew Valesano ◽  
William Fitzsimmons ◽  
Christopher Blair ◽  
Robert Woods ◽  
Julie Gilbert ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Panel Member ◽  
Illumina Miseq ◽  
Clinical Microbiology Laboratory ◽  
University Of Michigan ◽  
Viral Genomes ◽  
Genomic Epidemiology ◽  
Ann Arbor ◽  
Washtenaw County ◽  
The University

Abstract Background Understanding SARS-CoV-2 transmission dynamics is critical for controlling and preventing outbreaks. The genomic epidemiology of SARS-CoV-2 on college campuses has not been comprehensively studied, and the extent to which campus-associated outbreaks lead to transmission in nearby communities is unclear. We used high-density genomic surveillance to track SARS-CoV-2 transmission across the University of Michigan-Ann Arbor campus and Washtenaw County during the Fall 2020 semester. Methods We retrieved all available residual diagnostic specimens from the Michigan Medicine Clinical Microbiology Laboratory and University Health Service that were positive for SARS-CoV-2 from August 16th – November 25th, 2020 (n = 2245). We extracted viral RNA, amplified the SARS-CoV-2 genome by multiplex RT-PCR, and sequenced these amplicons on an Illumina MiSeq. We applied maximum likelihood phylogenetic analysis to whole genome sequences to define and characterize transmission lineages. Results We assembled complete viral genomes from 1659 individual infections, representing roughly 25% of confirmed cases in Washtenaw County across the fall semester. Of these cases, 468 were University of Michigan students. Phylogenetic analysis revealed 203 genetically distinct introductions of SARS-CoV-2 into the student population, most of which were singletons (n = 171) or small clusters of 2 – 8 students. We identified two large SARS-CoV-2 transmission lineages (115 and 73 students, respectively), including individuals from multiple on-campus residences. Viral descendants of these student outbreaks were rare, constituting less than 4% of cases in the community. Conclusion We identified many SARS-CoV-2 transmission introductions into the University of Michigan campus in Fall 2020. While there was widespread transmission among students, there is little evidence that these outbreaks significantly contributed to the rise in COVID-19 cases that Washtenaw County experienced in November 2020. Disclosures Adam Lauring, MD, PhD, Roche (Advisor or Review Panel member) Sanofi (Consultant)

scholarly journals Pigment production by Cryptococcus neoformans and other Cryptococcus species from aminophenols and diaminobenzenes

1978 ◽  
Vol 7 (2) ◽  
pp. 146-152
Author(s):  
S Chaskes ◽  
R L Tyndall
Keyword(s):  
Cryptococcus Neoformans ◽  
Species Group ◽  
Pigment Production ◽  
The Other ◽  
Aminosalicylic Acid ◽  
Pigmentation Pattern ◽  
Unique Pattern ◽  
Group I ◽  
Cryptococcus Species ◽  
Group Ii

Cryptococcus neoformans and other Cryptococcus species can produce pigment(s) from many aminophenol and diaminobenzene compounds. Pigment production from these compounds is similar to the conversion of diphenols to melanin by C. neoformans. Several pigmentation patterns (resulting in the identification or grouping of Cryptococcus species) have been observed by using diaminobenzene and aminophenol compounds as substrates. The most common pigmentation pattern observed was pigment production by both C. neoformans and C. terreus. In contrast to the diphenols, only two aminophenols (4-hydroxymetanilamide and 3-aminotyrosine) were found to be highly specific as substrates. They allowed only C. neoformans to produce pigment. When 4-aminosalicylic acid was the substrate, a unique pattern was observed because only C. terreus, C. diffluens, and C. albidus produced pigment. Finally, a pattern was observed in which C. neoformans produced large amounts of pigment from aminophenol and diaminobenzene compounds, whereas the other Cryptococcus species produced smaller amounts. A simplified scheme with three substrates resulted in the identification of C. terreus and C. neoformans as well as two groups of other Cryptococcus species, group I (C. albidus and C. diffluens) and group II (C. laurentii and C. luteolus).

scholarly journals Metagenomics Study of Fungi and Fungi-Like Organisms Associated With the Seagrass Halophila Stipulacea (Forssk.) Asch. From Al-Leith Mangroves, Saudi Arabia

2021 ◽  
Author(s):  
Mohamed Abdel-Wahab ◽  
Ali H. Bahkali ◽  
Abdallah M El-Gorban ◽  
Gareth Jones
Keyword(s):  
Saudi Arabia ◽  
Molecular Species ◽  
Illumina Miseq ◽  
Lsu Rdna ◽  
Variable Regions ◽  
Halophila Stipulacea ◽  
Three Samples ◽  
Total Dna ◽  
High Diversity ◽  
Sea Coast

Abstract This is the first metagenomics study of the fungal community associated with leaves of the seagrass Halophila stipulacea. Five leave samples were collected from the Al-Leith mangroves along the Red Sea coast of Saudi Arabia. Total DNA was extracted from 250 mg of each sample and the first 300 bp (contains D1-D2 variable regions) of the LSU rDNA amplicon was sequenced with the Illumina MiSeq (bTEFAP). A total of 928,626 reads were obtained from the five samples. The sequence reads belonged to the kingdoms: Metazoa (48.1% of the total reads), Viridiplantae (41.1%), Eukaryota (8.8%), Fungi (1.96%), Bacteria (0.09%), and Archaea (0.0001%). Fungi represented between 1.1% and 5.8% of the total reads in the five samples. A total of 18,279 reads (representing 1.96% of the total reads) were recorded from the 5 samples representing 296 molecular species (OTUs) that belong to 13 fungal phyla. At the phylum level, Basidiomycota dominated the community (37.2–51.6%) in three samples, while Neocallimastigomycota (37.5%) and Mucoromycota (42.1%) dominated the community in the fourth and the fifth sample respectively. High diversity of OTUs (28 molecular species) were recorded from the monokaryotic subkingdom with five unknown basal lineages that are not aligning with any known taxa. Total number of sequence reads of fungi-like organisms (Stramenopiles) from the five samples ranged between 0.16% of total reads in the fifth sample (AL-Hs05) to 2.9% in the first one (AL-Hs01). Majority of the fungi-like organisms reads (93.6%) belong to the phylum Oomycota, followed by Opisthokonts (Fungi/Metazoa group) representing 6.4% of fungi-like reads. Monokaryon phyla (i.e. Chytridiomycota, Mucoromycota and Neocallimastigomycota) and fungi-like organisms occupied a major portion of the sequences reads followed by Basidiomycota and Ascomycota. Our results support findings that the majority of fungi and fungi-like organisms’ communities are so far unknown with seven deep branching lineages remain to be cultivated.

scholarly journals Diversity of carbapenem-resistant Klebsiella pneumoniae ST14 and emergence of a subgroup with KL64 capsular locus in the Arabian Peninsula

2021 ◽  
Author(s):  
Shaimaa F. Mouftah ◽  
Tibor Pál ◽  
Paul G. Higgins ◽  
Akela Ghazawi ◽  
Youssef Idaghdour ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Klebsiella Pneumoniae ◽  
United Arab Emirates ◽  
Core Genome ◽  
Arabian Peninsula ◽  
Illumina Miseq ◽  
Carbapenem Resistant ◽  
Distinct Cluster ◽  
Recent Emergence ◽  
Miseq Platform

AbstractTo understand the reasons of successful spread of carbapenem-resistant Klebsiella pneumoniae ST14 (CRKP-ST14) in countries of the Arabian Peninsula, the resistome, capsular locus, carbapenemase carrying plasmid types, and core genome of isolates from the region were compared to global isolates. Thirty-nine CRKP-ST14 strains isolated from 13 hospitals in the United Arab Emirates, Bahrain, and Saudi Arabia were selected for whole genome sequencing on Illumina MiSeq platform based on the variety of carbapenemase genes carried and plasmids bearing these genes. Their resistome, capsular locus, and core genome MLST were compared to 173 CRKP-ST14 genomes available in public databases. The selected 39 CRKP-ST14 produced either NDM-1, OXA-48, OXA-162, OXA-232, KPC-2, or co-produced NDM-1 and an OXA-48-like carbapenemase. cgMLST revealed three clusters: 16 isolates from five UAE cities (C1), 11 isolates from three UAE cities and Bahrain (C2), and 5 isolates from Saudi Arabia (C3), respectively, and seven singletons. Resistance gene profile, carbapenemase genes, and their plasmid types were variable in both C1 and C2 clusters. The majority of CRKP-ST14 had KL2, but members of the C2 cluster and two further singletons possessed KL64 capsular locus. Based on cgMLST comparison of regional and global isolates, CRKP-ST14 with KL64 from four continents formed a distinct cluster, suggesting a recent emergence and spread of this variant. Our findings confirmed clonal transmission coupled with likely horizontal gene transfer in carbapenem-resistant Klebsiella pneumoniae ST14. Dissemination of this genetically flexible, highly resistant clone warrants further monitoring.

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