A target-specific assay for rapid and quantitative detection of Mycobacterium chimaera DNA in environmental and clinical specimens

ABSTRACT Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera . Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera . We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera . Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera .

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Activité antimicrobienne d'antioxydants phénoliques

Canadian Journal of Microbiology ◽

10.1139/m78-212 ◽

1978 ◽

Vol 24 (11) ◽

pp. 1306-1320 ◽

Cited By ~ 6

Author(s):

Pierre Turcotte ◽

Samir A. Saheb

Keyword(s):

Culture Media ◽

Osmotic Shock ◽

Bacterial Species ◽

Butylated Hydroxytoluene ◽

In Vitro Assays ◽

Gram Negative Bacteria ◽

Gram Negative ◽

The Family ◽

Increased Sensitivity

The antimicrobial activity of three antioxydants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and ethoxyquin (ETO) was studied. In vitro assays showed that when these antioxydants are added to the culture media at concentrations lower or equal to that used in nutrition, they inhibit or decrease the growth of certain microorganisms. BHT showed the most marked effect, affecting Gram-positive bacteria at a higher degree than the Gram-negative bacteria belonging to the family Enterobacteriaceae. Inactivation study of different bacterial species by BHT revealed differences in sensitivity among a single genus and between strains of the same species. The association of ETO with BHT results in an increase of the inhibitory activity. The increased sensitivity to BHT resulting from the osmotic shock of Escherichia coli cells suggests that the resistance to BHT of the Gram-negative bacteria belonging to the family Enterobacteriaceae might be due in part to the structure of their cell wall.

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SpeciesPrimer: A bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species

10.7287/peerj.preprints.27870 ◽

2019 ◽

Author(s):

Matthias Dreier ◽

Hélène Berthoud ◽

Noam Shani ◽

Daniel Wechsler ◽

Pilar Junier

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Bacterial Species ◽

Primer Design ◽

Diagnostic Methods ◽

Food Microbiology ◽

Bioinformatics Pipeline ◽

Specific Primers ◽

Species Specific

Background. Quantitative real-time PCR (qPCR) is a well-established method for detecting and quantifying bacteria, and it is progressively replacing culture-based diagnostic methods in food microbiology. High-throughput qPCR using microfluidics brings further advantages by providing faster results, decreasing the costs per sample and reducing errors due to automatic distribution of samples and reactants. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in a given system (for instance, cheese), the preliminary setup of qPCR assays working efficiently under identical PCR conditions is required. Identification of target-specific nucleotide sequences and design of specific primers are the most challenging steps in this process. To date, most available tools for primer design require either laborious manual manipulation or high-performance computing systems. Results. We developed the SpeciesPrimer pipeline for automated high-throughput screening of species-specific target regions and the design of dedicated primers. Using SpeciesPrimer specific primers were designed for four bacterial species of importance in cheese quality control, namely Enterococcus faecium, Enterococcus faecalis, Pediococcus acidilactici and Pediococcus pentosaceus. Selected primers were first evaluated in silico and subsequently in vitro using DNA from pure cultures of a variety of strains found in dairy products. Specific qPCR assays were developed and validated, satisfying the criteria of inclusivity, exclusivity and amplification efficiencies. Conclusion. In this work, we present the SpeciesPrimer pipeline, a tool to design species-specific primers for the detection and quantification of bacterial species. We use SpeciesPrimer to design qPCR assays for four bacterial species and describe a workflow to evaluate the designed primers. SpeciesPrimer facilitates efficient primer design for species-specific quantification, paving the way for a fast and accurate quantitative investigation of microbial communities.

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SpeciesPrimer: A bioinformatics pipeline dedicated to the design of qPCR primers for the quantification of bacterial species

10.7287/peerj.preprints.27870v1 ◽

2019 ◽

Author(s):

Matthias Dreier ◽

Hélène Berthoud ◽

Noam Shani ◽

Daniel Wechsler ◽

Pilar Junier

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Bacterial Species ◽

Primer Design ◽

Diagnostic Methods ◽

Food Microbiology ◽

Bioinformatics Pipeline ◽

Specific Primers ◽

Species Specific

Background. Quantitative real-time PCR (qPCR) is a well-established method for detecting and quantifying bacteria, and it is progressively replacing culture-based diagnostic methods in food microbiology. High-throughput qPCR using microfluidics brings further advantages by providing faster results, decreasing the costs per sample and reducing errors due to automatic distribution of samples and reactants. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in a given system (for instance, cheese), the preliminary setup of qPCR assays working efficiently under identical PCR conditions is required. Identification of target-specific nucleotide sequences and design of specific primers are the most challenging steps in this process. To date, most available tools for primer design require either laborious manual manipulation or high-performance computing systems. Results. We developed the SpeciesPrimer pipeline for automated high-throughput screening of species-specific target regions and the design of dedicated primers. Using SpeciesPrimer specific primers were designed for four bacterial species of importance in cheese quality control, namely Enterococcus faecium, Enterococcus faecalis, Pediococcus acidilactici and Pediococcus pentosaceus. Selected primers were first evaluated in silico and subsequently in vitro using DNA from pure cultures of a variety of strains found in dairy products. Specific qPCR assays were developed and validated, satisfying the criteria of inclusivity, exclusivity and amplification efficiencies. Conclusion. In this work, we present the SpeciesPrimer pipeline, a tool to design species-specific primers for the detection and quantification of bacterial species. We use SpeciesPrimer to design qPCR assays for four bacterial species and describe a workflow to evaluate the designed primers. SpeciesPrimer facilitates efficient primer design for species-specific quantification, paving the way for a fast and accurate quantitative investigation of microbial communities.

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The Circulating Cell-free Genome Atlas (CCGA) Study: Size selection of cell-free DNA (cfDNA) fragments.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.3103 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 3103-3103 ◽

Cited By ~ 1

Author(s):

Darya Filippova ◽

Matthew H. Larson ◽

M. Cyrus Maher ◽

Robert Calef ◽

Monica Pimentel ◽

...

Keyword(s):

In Silico ◽

Potential Effect ◽

Classification Performance ◽

Size Selection ◽

Increased Sensitivity ◽

Cancer Types ◽

N 93 ◽

Tumor Types ◽

Selection Of

3103 Background: Detection of somatic copy number aberrations in individuals with cancer via cfDNA whole-genome sequencing (WGS) is challenging at low tumor fractions. Given that tumor-derived cfDNA fragments are shorter than those from healthy tissues, this exploratory analysis evaluated the potential effect of size selection on the ability to detect cancer. Methods: CCGA WGS libraries were in silico and in vitro size selected to estimate the change in tumor fraction by tumor types (breast, lung, and colorectal [CRC]) and stage (I-III vs IV). In silico analyses used clinically evaluable training set samples with WGS assay results (n = 1422: 560 non-cancer [NC], 862 cancer [C] stages I-IV); classification (cancer/non-cancer) performance was estimated using fragments within the 90-150 bp range. In vitro analyses used a subset of samples (n = 93: 28 NC, 65 C stages I-IV), including C cases sampled within a range of tumor fractions; tumor fraction was also measured at each progressive removal of maximum-length fragments (intervals of 10 bp: 150 bp down to 50 bp). Results: In silico and in vitro analyses, respectively, resulted in median 2.00±0.58-fold (at 6.91±2.64X depth) and 2.00±0.52-fold (at 23±4.45X depth) increases, in overall tumor fraction (compared to non-size-selected 36X depth). This was consistent across tumor types ( in silico: 1.78±0.73 breast, 2.00±0.58 CRC, 2.00±0.41 lung; in vitro: 2.00±0.82 breast, 2.51±0.52 CRC, 2.53±0.94 lung) and stages ( in silico: 2.00±0.74 I-III, 1.78±0.52 IV; in vitro: 2.00±0.55 I-III, 1.68±0.29 IV). Tumor fraction increased with initial fragment length titrations, but not following size selection to shorter lengths ( < 140 bp). Classifier trained on in silico size-selected data had increased sensitivity at 98% specificity compared to those trained on non-size-selected data (p < 1e-5). Conclusions: In silico and in vitro size selection consistently increased tumor fraction across cancer types and stages, and this increase was maximized by tuning the length range of size selection. Relative to full-depth data, classification performance improved significantly. These data suggest that size selection targeting cfDNA under 140 bp may enhance cfDNA-based cancer detection. Clinical trial information: NCT02889978.

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Functional characterization of the Mycobacterium tuberculosis serine/threonine kinase PknJ

Microbiology ◽

10.1099/mic.0.038133-0 ◽

2010 ◽

Vol 156 (6) ◽

pp. 1619-1631 ◽

Cited By ~ 21

Author(s):

Jichan Jang ◽

Alexandre Stella ◽

Frédéric Boudou ◽

Florence Levillain ◽

Eliette Darthuy ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Transmembrane Domain ◽

Bacterial Species ◽

Functional Characterization ◽

Mycolic Acid ◽

Serine Threonine Kinase ◽

Environmental Signals ◽

Mycobacterial Genome ◽

Peptide Chip

Eukaryotic-like Ser/Thr protein kinases (STPKs) are present in many bacterial species, where they control various physiological and virulence processes by enabling microbial adaptation to specific environmental signals. PknJ is the only member of the 11 STPKs identified in Mycobacterium tuberculosis that still awaits characterization. Here we report that PknJ is a functional kinase that forms dimers in vitro, and contains a single transmembrane domain. Using a high-density peptide-chip-based technology, multiple potential mycobacterial targets were identified for PknJ. We confirmed PknJ-dependent phosphorylation of four of these targets: PknJ itself, which autophosphorylates at Thr168, Thr171 and Thr173 residues; the transcriptional regulator EmbR; the methyltransferase MmaA4/Hma involved in mycolic acid biosynthesis; and the dipeptidase PepE, whose encoding gene is located next to pknJ in the mycobacterial genome. Our results provide a number of candidate phospho-targets for PknJ and possibly other mycobacterial STPKs that could be studied to investigate the role of STPKs in M. tuberculosis physiology and virulence.

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Streptococcus pneumoniae Modulates Staphylococcus aureus Biofilm Dispersion and the Transition from Colonization to Invasive Disease

mBio ◽

10.1128/mbio.02089-17 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 12

Author(s):

Ryan M. Reddinger ◽

Nicole R. Luke-Marshall ◽

Shauna L. Sauberan ◽

Anders P. Hakansson ◽

Anthony A. Campagnari

Keyword(s):

Staphylococcus Aureus ◽

Streptococcus Pneumoniae ◽

Influenza A Virus ◽

Bacterial Pneumonia ◽

Influenza A ◽

Bacterial Species ◽

Invasive Disease ◽

Secondary Bacterial Pneumonia

ABSTRACTStreptococcus pneumoniaeandStaphylococcus aureusare ubiquitous upper respiratory opportunistic pathogens. Individually, these Gram-positive microbes are two of the most common causative agents of secondary bacterial pneumonia following influenza A virus infection, and they constitute a significant source of morbidity and mortality. Since the introduction of the pneumococcal conjugate vaccine, rates of cocolonization with both of these bacterial species have increased, despite the traditional view that they are antagonistic and mutually exclusive. The interactions betweenS. pneumoniaeandS. aureusin the context of colonization and the transition to invasive disease have not been characterized. In this report, we show thatS. pneumoniaeandS. aureusform stable dual-species biofilms on epithelial cellsin vitro. When these biofilms are exposed to physiological changes associated with viral infection,S. pneumoniaedisperses from the biofilm, whereasS. aureusdispersal is inhibited. These findings were supported by results of anin vivostudy in which we used a novel mouse cocolonization model. In these experiments, mice cocolonized in the nares with both bacterial species were subsequently infected with influenza A virus. The coinfected mice almost exclusively developed pneumococcal pneumonia. These results indicate that despite our previous report thatS. aureusdisseminates into the lungs of mice stably colonized with these bacteria following influenza A virus infection, cocolonization withS. pneumoniae in vitroandin vivoinhibitsS. aureusdispersal and transition to disease. This study provides novel insight into both the interactions betweenS. pneumoniaeandS. aureusduring carriage and the transition from colonization to secondary bacterial pneumonia.IMPORTANCEIn this study, we demonstrate thatStreptococcus pneumoniaecan modulate the pathogenic potential ofStaphylococcus aureusin a model of secondary bacterial pneumonia. We report that host physiological signals related to viral infection cease to elicit a dispersal response fromS. aureuswhile in a dual-species setting withS. pneumoniae, in direct contrast to results of previous studies with each species individually. This study underscores the importance of studying polymicrobial communities and their implications in disease states.

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Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657214 ◽

1982 ◽

Vol 48 (01) ◽

pp. 049-053 ◽

Cited By ~ 47

Author(s):

C G Fenn ◽

J M Littleton

Keyword(s):

Platelet Aggregation ◽

Lipid Composition ◽

Saturated Fatty Acids ◽

Calcium Ionophore ◽

Cholesterol Content ◽

Membrane Lipid ◽

Calcium Ionophore A23187 ◽

Ionophore A23187 ◽

Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

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Diagnostic methods for confirming drug-allergy – the lymphocyte transformation test in dermatology

Orvosi Hetilap ◽

10.1556/oh.2008.28215 ◽

2008 ◽

Vol 149 (24) ◽

pp. 1107-1114

Author(s):

Sarolta Makó ◽

Réka Lepesi-Benkő ◽

Márta Marschalkó ◽

Gyöngyvér Soós ◽

Sarolta Kárpáti

Keyword(s):

Drug Allergy ◽

Lymphocyte Transformation ◽

Lymphocyte Transformation Test ◽

Diagnostic Methods

A gyógyszermellékhatások felismerése és a tüneteket kiváltó gyógyszer oki szerepének bizonyítása komoly felkészültséget igényel. E közlemény célja a gyógyszerallergiás reakciók diagnosztikai lehetőségeinek rövid áttekintése és a lymphocytatranszformációs teszt gyógyszer-hiperszenzitivitási reakciókban való bizonyító szerepének bőrgyógyászati szempontok szerinti értékelése. A lymphocytatranszformációs teszt azon a megfigyelésen alapul, hogy a gyógyszerrel való első találkozáskor kialakult antigénspecifikus T-sejtek osztódni kezdenek az antigénnel való in vitro megismételt találkozás után. A szenzibilizációt az osztódó T-sejtekbe történő 3 H-timidin-beépülés mértéke jelzi. A hatóanyag-specifikus T-sejtek szinte mindig részt vesznek a gyógyszerallergiás reakciókban, ezért a vizsgálat előnye, hogy sok gyógyszernél és különböző immunreakciók eseteiben egyaránt jól alkalmazható. Hátránya a munkaigényesség, valamint az, hogy specificitásának és szenzitivitásának bizonyításához hiányoznak a széles körű, nagy beteganyagon elvégzett tanulmányok. Emiatt a teszt nem egyértelműen elfogadott a gyógyszerallergia igazolására. Hiányosságai ellenére azonban, jobb prediktív értékű egyéb vizsgálatok hiányában, a lymphocytatranszformációs tesztnek fontos szerepe van a gyógyszerallergiák diagnosztizálása terén.

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