scholarly journals Accuracy of an amplicon-sequencing nanopore approach to identify variants in tuberculosis drug-resistance-associated genes

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Carla Mariner-Llicer ◽  
Galo A. Goig ◽  
Laura Zaragoza-Infante ◽  
Manuela Torres-Puente ◽  
Luis Villamayor ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Type Species ◽  
Variant Calling ◽  
Targeted Sequencing ◽  
Clinical Samples ◽  
Drug Resistant ◽  
Content Type ◽  
Link Type ◽  
Resistant Strains ◽  
Drug Resistant Strains

A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis , identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of M. tuberculosis using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study’s main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results.

scholarly journals Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples

2013 ◽  
Vol 63 (Pt_4) ◽  
pp. 1323-1328 ◽  
Author(s):  
William J. Wolfgang ◽  
Teresa V. Passaretti ◽  
Reashma Jose ◽  
Jocelyn Cole ◽  
An Coorevits ◽  
...  
Keyword(s):  
Type Strain ◽  
Type Species ◽  
Novel Species ◽  
Sequence Similarity ◽  
23S Rrna ◽  
Clinical Samples ◽  
Rrna Gene ◽  
The Novel ◽  
Content Type ◽  
Link Type

A polyphasic analysis was undertaken of seven independent isolates of Gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7–100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA–DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbour, N. lactamica . Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the novel species belongs in the genus Neisseria . The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The cellular fatty acid profile, together with other phenotypic characters, further supports the inclusion of the novel species in the genus Neisseria . The name Neisseria oralis sp. nov. (type strain 6332T  = DSM 25276T  = LMG 26725T) is proposed.

scholarly journals In Vitro Activity and MIC of Sitafloxacin against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis Isolated in Thailand

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Manoon Leechawengwongs ◽  
Therdsak Prammananan ◽  
Sarinya Jaitrong ◽  
Pamaree Billamas ◽  
Nampueng Makhao ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Resistant Strains ◽  
Drug Resistant Strains

ABSTRACT New fluoroquinolones (FQs) have been shown to be more active against drug-resistant Mycobacterium tuberculosis strains than early FQs, such as ofloxacin. Sitafloxacin (STFX) is a new fluoroquinolone with in vitro activity against a broad range of bacteria, including M. tuberculosis. This study aimed to determine the in vitro activity of STFX against all groups of drug-resistant strains, including multidrug-resistant M. tuberculosis (MDR M. tuberculosis), MDR M. tuberculosis with quinolone resistance (pre-XDR), and extensively drug-resistant (XDR) strains. A total of 374 drug-resistant M. tuberculosis strains were tested for drug susceptibility by the conventional proportion method, and 95 strains were randomly submitted for MIC determination using the microplate alamarBlue assay (MABA). The results revealed that all the drug-resistant strains were susceptible to STFX at a critical concentration of 2 μg/ml. Determination of the MIC90s of the strains showed different MIC levels; MDR M. tuberculosis strains had a MIC90 of 0.0625 μg/ml, whereas pre-XDR and XDR M. tuberculosis strains had identical MIC90s of 0.5 μg/ml. Common mutations within the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB did not confer resistance to STFX, except that double mutations of GyrA at Ala90Val and Asp94Ala were found in strains with a MIC of 1.0 μg/ml. The results indicated that STFX had potent in vitro activity against all the groups of drug-resistant M. tuberculosis strains and should be considered a new repurposed drug for treatment of multidrug-resistant and extensively drug-resistant TB.

Kerstersia similis sp. nov., isolated from human clinical samples

2012 ◽  
Vol 62 (Pt_9) ◽  
pp. 2156-2159 ◽  
Author(s):  
Peter Vandamme ◽  
Evie De Brandt ◽  
Kurt Houf ◽  
Thierry De Baere
Keyword(s):  
Type Strain ◽  
Type Species ◽  
Gene Sequence ◽  
Clinical Samples ◽  
Content Type ◽  
Biochemical Characteristics ◽  
Link Type ◽  
Gene Sequence Analysis ◽  
The Usa ◽  
Kerstersia Gyiorum

Analysis of gyrB gene sequences, (GTG)5-primed PCR fingerprinting and biochemical characteristics determined in the Biolog GEN III microtest system were used to differentiate an unnamed Kerstersia species from Kerstersia gyiorum , the type and only named species in this genus. The inability to oxidize d-galacturonic and d-glucuronic acids and the ability to oxidize d-serine, along with gyrB gene sequence analysis and (GTG)5-PCR fingerprints, readily differentiated the unnamed taxon from the type species. Therefore, we propose to formally classify this unnamed taxon as Kerstersia similis sp. nov. with strain LMG 5890T ( = CCUG 46999T), isolated from a leg wound in the USA in 1983, as the type strain.

scholarly journals Sontochin as a Guide to the Development of Drugs against Chloroquine-Resistant Malaria

2012 ◽  
Vol 56 (7) ◽  
pp. 3475-3480 ◽  
Author(s):  
Sovitj Pou ◽  
Rolf W. Winter ◽  
Aaron Nilsen ◽  
Jane Xu Kelly ◽  
Yuexin Li ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Plasmodium Yoelii ◽  
Significant Activity ◽  
Drug Resistant ◽  
Content Type ◽  
Resistant Strains ◽  
Drug Resistant Strains ◽  
Derivatives Of

ABSTRACTSontochin was the original chloroquine replacement drug, arising from research by Hans Andersag 2 years after chloroquine (known as “resochin” at the time) had been shelved due to the mistaken perception that it was too toxic for human use. We were surprised to find that sontochin, i.e., 3-methyl-chloroquine, retains significant activity against chloroquine-resistant strains ofPlasmodium falciparum in vitro. We prepared derivatives of sontochin, “pharmachins,” with alkyl or aryl substituents at the 3 position and with alterations to the 4-position side chain to enhance activity against drug-resistant strains. Modified with an aryl substituent in the 3 position of the 7-chloro-quinoline ring, Pharmachin 203 (PH-203) exhibits low-nanomolar 50% inhibitory concentrations (IC50s) against drug-sensitive and multidrug-resistant strains andin vivoefficacy against patent infections ofPlasmodium yoeliiin mice that is superior to chloroquine. Our findings suggest that novel 3-position aryl pharmachin derivatives have the potential for use in treating drug resistant malaria.

scholarly journals Use of the NP-40 Detergent-Mediated Assay in Discovery of Inhibitors of β-Hematin Crystallization

2011 ◽  
Vol 55 (7) ◽  
pp. 3363-3369 ◽  
Author(s):  
Rebecca D. Sandlin ◽  
Melissa D. Carter ◽  
Patricia J. Lee ◽  
Jennifer M. Auschwitz ◽  
Susan E. Leed ◽  
...  
Keyword(s):  
Drug Target ◽  
Inhibitory Concentration ◽  
Protozoan Parasite ◽  
Drug Resistant ◽  
Digestive Vacuole ◽  
Content Type ◽  
Parasite Survival ◽  
Resistant Strains ◽  
Drug Resistant Strains ◽  
Important Drug

ABSTRACTThe protozoan parasite responsible for malaria affects over 500 million people each year. Current antimalarials have experienced decreased efficacy due to the development of drug-resistant strains ofPlasmodiumspp., resulting in a critical need for the discovery of new antimalarials. Hemozoin, a crystalline by-product of heme detoxification that is necessary for parasite survival, serves as an important drug target. The quinoline antimalarials, including amodiaquine and chloroquine, act by inhibiting the formation of hemozoin. The formation of this crystal does not occur spontaneously, and recent evidence suggests crystallization occurs in the presence of neutral lipid particles located in the acidic digestive vacuole of the parasite. To mimic these conditions, the lipophilic detergent NP-40 has previously been shown to successfully mediate the formation of β-hematin, synthetic hemozoin. Here, an NP-40 detergent-based assay was successfully adapted for use as a high-throughput screen to identify inhibitors of β-hematin formation. The resulting assay exhibited a favorableZ′ of 0.82 and maximal drift of less than 4%. The assay was used in a pilot screen of 38,400 diverse compounds at a screening concentration of 19.3 μM, resulting in the identification of 161 previously unreported β-hematin inhibitors. Of these, 48 also exhibited ≥90% inhibition of parasitemia in aPlasmodium falciparumwhole-cell assay at a screening concentration of 23 μM. Eight of these compounds were identified to have nanomolar 50% inhibitory concentration values near that of chloroquine in this assay.

scholarly journals An antipathogenic compound that targets the OxyR peroxide sensor in Pseudomonas aeruginosa

2021 ◽  
Vol 70 (4) ◽  
Author(s):  
Hyo-Young Oh ◽  
Shivakumar S. Jalde ◽  
In-Young Chung ◽  
Yeon-Ji Yoo ◽  
Hye-Jeong Jang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Resistance ◽  
Stress Responses ◽  
Type Species ◽  
Reduced Form ◽  
Infection Model ◽  
Redox Cycle ◽  
Content Type ◽  
Link Type ◽  
Computer Based

Introduction. Antipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance. Hypothesis/Gap Statment. Peroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy. Aim. This study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa . Methodology. Computer-based virtual screening under consideration of the ‘eNTRy’ rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR. Results. We first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H2O2 treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus . Conclusion. These results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.

scholarly journals Genetic diversity and epidemiology of accessory gene regulator loci in Clostridioides difficile

2020 ◽  
Vol 2 (7) ◽  
Author(s):  
Yuta Okada ◽  
Shu Okugawa ◽  
Mahoko Ikeda ◽  
Tatsuya Kobayashi ◽  
Ryoichi Saito ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
In Silico ◽  
Type Species ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Accessory Gene ◽  
Content Type ◽  
Link Type ◽  
Accessory Gene Regulator ◽  
Clostridioides Difficile

Quorum sensing is known to regulate bacterial virulence, and the accessory gene regulator (agr) loci is one of the genetic loci responsible for its regulation. Recent reports examining Clostridioides difficile show that two agr loci, agr1 and agr2, regulate toxin production, but the diversity of agr loci and their epidemiology is unknown. In our study, in silico analysis was performed to research genetic diversity of agr, and C. difficile isolates from clinical samples underwent multilocus sequence typing (MLST) and PCR analysis of agr loci. To reveal the distribution of agr among different strains, phylogenetic analysis was also performed. In our in silico analysis, two different subtypes, named agr2R and agr2M, were found in agr2, which were previously reported. PCR analysis of 133 C . difficile isolates showed that 131 strains had agr1, 61 strains had agr2R, and 26 strains had agr2M; agr2R was mainly found in clade 1 or clade 2 organisms, whereas agr2M was only found in clade 4. With rare exception, agr1-negative sequence types (STs) belonged to clade C-Ⅰ and C-Ⅲ, and one clade 4 strain had agr2R. Our study revealed subtypes of agr2 not previously recognized, and the distribution of several agr loci in C. difficile . These findings provide a foundation for further functional and clinical research of the agr loci.

scholarly journals Snapshot of Mycobacterium tuberculosis Phylogenetics from an Indian State of Arunachal Pradesh Bordering China

2021 ◽  
Author(s):  
Rashmi S Mudliar ◽  
Umay Kulsum ◽  
Syed Beenish Rufai ◽  
Mika Umpo ◽  
Moi Nyori ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Drug Resistant ◽  
Second Line ◽  
Arunachal Pradesh ◽  
Tuberculosis Programme ◽  
Indian State ◽  
Northeastern State ◽  
Resistant Strains ◽  
Drug Resistant Strains

Uncontrolled transmission of Mycobacterium tuberculosis (M. tuberculosis, MTB) drug resistant strains is a challenge to control efforts of global tuberculosis programme. Due to increasing multi-drug resistant (MDR) cases in Arunachal Pradesh, a northeastern state of India, the tracking and tracing of these resistant MTB strains is crucial for infection control and spread of drug resistance. This study aims to correlate the phenotypic DST, genomic DST (gDST) and phylogenetic analysis of MDR-MTB strains in the region. Of total 200 suspected MDR-MTB isolates, 125(62.5%) were identified as MTB. MGIT-960 SIRE DST detected 71/125(56.8%) isolates as MDR/RR-MTB of which 22(30.9%) were detected resistant to second line drugs. Whole genome sequencing of 65 isolates and their gDST found Ser315Thr mutation in katG (35/45;77.8%) and Ser531Leu mutation in rpoB (21/41;51.2%) associated with drug resistance. SNP barcoding categorized the dataset with Lineage2 (41;63.1%) being predominant followed by Lineage3 (10;15.4%), Lineage1 (8;12.3%) and Lineage4 (6;9.2%) respectively. Phylogenetic assignment by cgMLST gave insights of two Beijing sub-lineages viz; 2.2.1 (SNP difference < 19) and 2.2.1.2 (SNP difference < 9) associated with recent ongoing transmission in Arunachal Pradesh. This study provides first insight in identifying the ongoing transmission of two virulent Beijing sub-lineages associated with TB drug resistance.

scholarly journals Role of Alanine Racemase Mutations in Mycobacterium tuberculosisd-Cycloserine Resistance

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Yoshio Nakatani ◽  
Helen K. Opel-Reading ◽  
Matthias Merker ◽  
Diana Machado ◽  
Sönke Andres ◽  
...  
Keyword(s):  
Alanine Racemase ◽  
Drug Resistant ◽  
Evolutionary Patterns ◽  
Content Type ◽  
Direct Measurements ◽  
Resistant Strains ◽  
Selection For ◽  
Drug Resistant Strains

ABSTRACT A screening of more than 1,500 drug-resistant strains of Mycobacterium tuberculosis revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, in vitro MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to d-cycloserine.

Activities of tubercular inflammation (on the morphological data) with different duration in the patients with drug-resistant pulmonary tuberculosis

2016 ◽  
Vol 10 (1) ◽  
pp. 0-0
Author(s):  
Елипашев ◽  
A. Elipashev ◽  
Никольский ◽  
V. Nikolskiy ◽  
Шпрыков ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Sensitivity ◽  
Morphological Data ◽  
Control Group ◽  
Drug Resistant ◽  
Specific Therapy ◽  
Resistant Tuberculosis ◽  
Number Of Patients ◽  
Resistant Strains ◽  
Drug Resistant Strains

The purpose of this research was to determine the dependence of the tubercular inflammation activity of varying duration of the disease and drug resistance. Morphological activity of inflammation in 161 patients with drug-resistant and 149 patients with retaining its high sensitivity was studied. Morphological assessment of the activity of specific changes in tuberculosis was carried out according to the B.M. Ariel classification (1998). It was revealed at morphologic study of resection material that the greatest activity of specific inflammation and its prevalence outside the main lesion was in the group of patients limited with drug resistance tuberculosis. It was noted the prevalence of IV-V degree of morphological activity of tubercular process in the study group by 3 times over the control group with disease duration of more than 1 year. Predominance of widespread active specific changes (IV degree) was determined in 2 times for the first educed patients of basic group with drug-resistant above a control group. This is due to increasing the number of patients with cavernous and fibro-cavernous tuberculosis.Thus, it is necessary to operate patients with drug-resistant tuberculosis as soon as possi-ble after adequate specific therapy and the presence of the signs of stabilization process, because as the full stabilization of tuberculosis process did not achieve according to the morphological study of surgical specimens in the preoperative period. Further specific therapy becomes futile due to the rise of drug resistance, the emer-gence of new drug-resistant strains of Mycobacterium tuberculosis.

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