scholarly journals Microscopy, Culture, and Quantitative Real-Time PCR Examination Confirm Internalization of Mycobacteria in Plants

2014 ◽  
Vol 80 (13) ◽  
pp. 3888-3894 ◽  
Author(s):  
M. Kaevska ◽  
S. Lvoncik ◽  
I. Slana ◽  
P. Kulich ◽  
P. Kralik
Keyword(s):  
Real Time ◽  
Plant Tissue ◽  
Culture Medium ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Source Of Infection ◽  
Different Types ◽  
Long Time ◽  
Different Strains

ABSTRACTThe environment is a reservoir of nontuberculous mycobacteria and is considered a source of infection for animals and humans. Mycobacteria can persist in different types of environments for a relatively long time. We have studied their possible internalization into plant tissue through intact, as well as damaged, root systems of different types of plants grownin vitroand under field conditions. The substrate into which plants were seeded was previously contaminated with different strains ofMycobacterium avium(108to 1010cells/g of soil) and feces from animals with paratuberculosis. We detectedM. aviumsubsp.avium,hominissuis, andparatuberculosisin the stems and leaves of the plants by both culture and real-time quantitative PCR. The presence of mycobacteria in the plant tissues was confirmed by microscopy. The concentration of mycobacteria found inside plant tissue was several orders of magnitude lower (up to 104cells/g of tissue) than the initial concentration of mycobacteria present in the culture medium or substrate. These findings led us to the hypothesis that plants may play a role in the spread and transmission of mycobacteria to other organisms in the environment.

scholarly journals Effects of antibiotics on Chlamydia trachomatis viability as determined by real-time quantitative PCR

2011 ◽  
Vol 60 (4) ◽  
pp. 508-514 ◽  
Author(s):  
Olivia Peuchant ◽  
Jean Philippe Duvert ◽  
Maïthé Clerc ◽  
Sophie Raherison ◽  
Christiane Bébéar ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Real Time ◽  
Dna Quantification ◽  
Mrna Transcription ◽  
Real Time Quantitative Pcr ◽  
Dna And Rna ◽  
Rna Quantification ◽  
Additional Cell

The objective of this study was to determine the effect of antibiotics on Chlamydia trachomatis viability by using a quantitative real-time PCR assay that measured DNA replication and mRNA transcription of the structural omp1 and omp2 genes, 16S rRNA and the groEL1 gene with and without antibiotics. Ofloxacin, moxifloxacin, azithromycin and doxycycline were tested against the serovar D and L2 reference strains and a derivative mutant resistant to fluoroquinolones, L2-OFXR, obtained by in vitro selection. Using DNA quantification, the antibiotic MIC was calculated when the number of DNA copies was equal to that of the chlamydial inoculum at time zero. This method allowed the easy determination of MICs by DNA quantification of the four selected genes and gave similar results to those obtained by immunofluorescence staining without biased interpretation. By using cDNA quantification, the lowest antibiotic concentration for which no RNA was transcribed corresponded to the minimum bactericidal concentration. C. trachomatis still transcribed the16S rRNA and groEL1 genes, even at concentrations well above the MIC, showing a bacteriostatic effect for all antibiotics tested. This method allows the study of antibiotic activity on growth and viability of C. trachomatis by DNA and RNA quantification at the same time without additional cell-culture passaging.

scholarly journals Assessment of the Kinetics of Treponema pallidum Dissemination into Blood and Tissues in Experimental Syphilis by Real-Time Quantitative PCR

2007 ◽  
Vol 75 (6) ◽  
pp. 2954-2958 ◽  
Author(s):  
Juan C. Salazar ◽  
Asha Rathi ◽  
Nelson L. Michael ◽  
Justin D. Radolf ◽  
Linda L. Jagodzinski
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Treponema Pallidum ◽  
Blood Components ◽  
Peripheral Blood Mononuclear ◽  
Real Time Quantitative Pcr ◽  
Kinetics Of

ABSTRACT Little is known about the size and kinetics of treponemal burdens in blood and tissues during acquired or experimental syphilitic infection. We used real-time quantitative PCR to measure Treponema pallidum DNA levels in rabbits infected intratesticularly with the prototype Nichols strain. At the outset, we performed a series of in vitro blood spiking experiments to determine the effect of blood processing procedures on the distribution of treponemes in various blood components. T. pallidum DNA levels in plasma and whole blood were approximately 10-fold higher than those in serum and more than 200-fold greater than those in peripheral blood mononuclear cells (PBMCs). Ten rabbits were inoculated intratesticularly with doses of treponemes ranging from 4 × 107 to 2 × 108 organisms. In five rabbits, T. pallidum DNA levels were measured sequentially in serum, plasma, whole blood, and PBMCs until sacrifice at peak orchitis, at which time brain, kidney, liver, spleen, and testicles were harvested; blood and organs were also harvested at orchitis from the other five rabbits. T. pallidum DNA was detected in plasma within 24 h postinfection. Treponeme levels in whole blood and blood components increased significantly with the development of peak orchitis. Overall, levels in serum and PBMCs were lower than those in plasma and whole blood; this disparity was particularly marked at early time points. Significantly greater numbers of spirochetes were found in the spleen than in liver, kidney, or brain tissue at the time of sacrifice. Our findings highlight the remarkable capacity of T. pallidum to disseminate from the site of infection to blood and tissues, and they identify the spleen as a prime target for treponemal invasion.

scholarly journals Molecular Tests That Target the RTX Locus Do Not Distinguish between Kingella kingae and the Recently Described Kingella negevensis Species

2017 ◽  
Vol 55 (10) ◽  
pp. 3113-3122 ◽  
Author(s):  
Nawal El Houmami ◽  
Janek Bzdrenga ◽  
Guillaume André Durand ◽  
Philippe Minodier ◽  
Hervé Seligmann ◽  
...  
Keyword(s):  
Rrna Gene ◽  
Invasive Infection ◽  
Pcr Assay ◽  
Kingella Kingae ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Molecular Tests ◽  
Invasive Infections ◽  
Culture Negative

ABSTRACTKingella kingaeis an important invasive pathogen in early childhood. The organism elaborates an RTX toxin presumably restricted to this species. Consequently, real-time quantitative PCR (qPCR) assays targeting the RTX locus have been developed in recent years and are gaining increasing use for the molecular diagnosis ofK. kingaeinfections. However, the present study shows thatKingella negevensis, aKingellaspecies newly identified in young children, harbors an identicalKingellaRTX locus, raising the question of whetherK. negevensiscan be misidentified asK. kingaeby clinical microbiology laboratories.In silicocomparison ofKingellasp. RTX andgroELgenes andin vitrostudies provided evidence that targeting thertxAandrtxBgenes could not differentiate between strains ofK. kingaeandK. negevensis, whereas targeting thegroELgene could. This prompted the design of a highly specific and sensitive qPCR assay targetingK. negevensis groEL(kngroEL). Ninety-nine culture-negative osteoarticular specimens from 99 children younger than 4 years of age were tested with a conventional 16S rRNA gene-based broad-range PCR assay andKingella-specificrtxB,K. kingae-specificgroEL(kkgroEL), andkngroELqPCR assays. Forty-two specimens werertxBpositive, including 41 that were alsokkgroELpositive and 1 (the remaining one) that waskngroELpositive. Thus, this study discloses an invasive infection caused byK. negevensisin humans and demonstrates that targeting the RTX locus cannot be used for the formal diagnosis ofK. kingaeinfections. These findings stress the need for further studies on the epidemiology of asymptomatic carriage and invasive infections caused byK. negevensisin humans.

scholarly journals Development of a Rapid and Sensitive Method Combining a Cellulose Ester Microfilter and a Real-Time Quantitative PCR Assay To Detect Campylobacter jejuni and Campylobacter coli in 20 Liters of Drinking Water or Low-Turbidity Waters

2011 ◽  
Vol 78 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Adeline Tissier ◽  
Martine Denis ◽  
Philippe Hartemann ◽  
Benoît Gassilloud
Keyword(s):  
Drinking Water ◽  
Real Time ◽  
Quantitative Pcr ◽  
Campylobacter Jejuni ◽  
Tap Water ◽  
Culture Method ◽  
Cellulose Ester ◽  
Campylobacter Coli ◽  
Content Type ◽  
Real Time Quantitative Pcr

ABSTRACTInvestigations ofCampylobacter jejuniandCampylobacter coliin samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection ofCampylobacterin 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) ofCampylobacterper 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with aC. colistrain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence ofC. jejuniandC. coligenomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination byCampylobacter.

scholarly journals Callus Induction and Shoot Formation for Mexican Red Bean (Phaseolus vulgaris L.) Pinto Cultivar in Vitro

2020 ◽  
pp. 1887-1893
Author(s):  
Rasha K. Mohammed Al-Saedi ◽  
Ansam G. Abdulhalem
Keyword(s):  
Growth Regulators ◽  
Culture Medium ◽  
Shoot Formation ◽  
Naphthalene Acetic Acid ◽  
Ms Medium ◽  
Phaseolus Vulgaris L ◽  
Different Types ◽  
Red Bean ◽  
Internode Explants

     The current study aimed to adopt a method for inducing callus cells and regenerating the important common red bean using different types of growth regulators such as N6-benzylaminopurine (BAP), Naphthalene acetic acid (NAA), and Thidiazuron (TDZ). Different types of common bean pinto cultivar explants, such as  internodes, cotyledons and roots,  were inoculated on Murashige and Skoog medium (MS) provided with different combinations of plant growth regulators, including 1- BAP (5 mg/l) 2-BAP (4.5 mg/l) NAA (0.5 mg/l), 3- BAP (4.5 mg/l), and TDZ (0.1mg/l). Callus was initiated on MS culture medium supplied with 5 mg/l BAP for all explants (internodes, cotyledons, and roots) at 50, 20, and 10% respectively, while adding NAA with 0.5mg/l showed a low percentage of callus (30%) only in the internode explants. Optimum results were obtained by growing the internodes on MS medium with 4.5 mg/l BA and either 0.5 mg/l NAA or 0.1 mg/l TDZ, transplanting the derived shoots into internodes and cotyledons with 70 and 10% respectively. This study concludes that the internodes as explants have the best growth results.

WHEAT ANDROCLINIC CALLI: DATA OF SCANNING ELECTRONIC MICROSCOPY

2021 ◽  
Vol 0 (4) ◽  
pp. 41-47
Author(s):  
O.A. SELDIMIROVA ◽  
Keyword(s):  
Culture Medium ◽  
Scanning Electronic Microscopy ◽  
Electronic Microscopy ◽  
Spring Soft Wheat ◽  
Fundamental Possibility ◽  
Morphological Status ◽  
Different Types ◽  
Morphogenesis In Vitro ◽  
Morphogenic Callus

The processes of formation different types of calli, as well as the morphogenesis pathways in morphogenic calli, were studied by scanning electron microscopy (SEM) during anther culture in vitro in hybrid line Fotos of spring soft wheat. The microspore haploid origin of calli has been proven. The morphological status of the obtained calli was determined. It was shown that morphogenic callus consists of small densely packed meristematic cells covered with extracellular substance. This type of calli was obtained using a variant of the Potato II induction culture medium, added by 1.0 mg/l synthetic auxin 2,4-D. Nonmorphogenic callus consists of large, elongated, loosely located cells with a smooth surface. This type of calli was obtained using a variant of the Potato II culture medium, added by 2.0 mg/l 2,4-D. It was found that the introduction of various IAA concentrations into the Blaydes nutrient medium for regeneration in morphogenic calli implements the following pathways of morphogenesis in vitro: embryoidogenesis (without IAA addition), gemmorhizogenesis (0.5 mg/l), and rhizogenesis (1.5 mg/l). Revealed degenerative changes in cells of nonmorphogenic calli. The fundamental possibility of regulating of the morphogenesis pathways of in vitro of morphogenic calli in the direction necessary for research in biotechnological research has been confirmed.

scholarly journals Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

2013 ◽  
Vol 57 (6) ◽  
pp. 2768-2779 ◽  
Author(s):  
Darren J. Creek ◽  
Brunda Nijagal ◽  
Dong-Hyun Kim ◽  
Federico Rojas ◽  
Keith R. Matthews ◽  
...  
Keyword(s):  
Cell Culture ◽  
Trypanosoma Brucei ◽  
Culture Medium ◽  
Culture Media ◽  
Culture Methods ◽  
Content Type ◽  
Drug Activity ◽  
Rational Development ◽  
High Concentrations

ABSTRACTIn vitroculture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream formTrypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supportsin vitrogrowth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.

In vitro biomechanical investigation of the stability and stress-shielding effect of lumbar interbody fusion devices

2000 ◽  
Vol 93 (2) ◽  
pp. 259-265 ◽  
Author(s):  
Masahiro Kanayama ◽  
Bryan W. Cunningham ◽  
Charles J. Haggerty ◽  
Kuniyoshi Abumi ◽  
Kiyoshi Kaneda ◽  
...  
Keyword(s):  
Interbody Fusion ◽  
Stress Shielding ◽  
Shielding Effect ◽  
Lumbar Interbody Fusion ◽  
Content Type ◽  
Different Types ◽  
Spine Model ◽  
Structural Allografts ◽  
Fine Print

Object. Interbody fusion devices are rapidly gaining acceptance as a method of ensuring lumbar interbody arthrodesis. Although different types of devices have been developed, the comparative reconstruction stability remains controversial. It also remains unclear how different stress-shielded environments are created within the devices. Using a calf spine model, this study was designed to compare the construct stiffness afforded by 11 differently designed lumbar interbody fusion devices and to quantify their stress-shielding effects by measuring pressure within the devices. Methods. Sixty-six lumbar specimens obtained from calves were subjected to anterior interbody reconstruction at L4–5 by using one of the following interbody fusion devices: four different threaded fusion cages (BAK device, BAK Proximity, Ray TFC, and Danek TIBFD), five different nonthreaded fusion devices (oval and circular Harms cages, Brantigan PLIF and ALIF cages, and InFix device); two different types of allograft (femoral ring and bone dowel) were used. Construct stiffness was evaluated in axial compression, torsion, flexion, and lateral bending. Prior to testing, a silicon elastomer was injected into the cages and intracage pressures were measured using pressure needle transducers. Conclusions. No statistical differences were observed in construct stiffness among the threaded cages and nonthreaded devices in most of the testing modalities. Threaded fusion cages demonstrated significantly lower intracage pressures compared with nonthreaded cages and structural allografts. Compared with nonthreaded cages and structural allografts, threaded fusion cages afforded equivalent reconstruction stiffness but provided more stress-shielded environment within the devices.

Restoration of virulence of axenically cultivated Entamoeba histolytica by cholesterol

1978 ◽  
Vol 24 (1) ◽  
pp. 63-65 ◽  
Author(s):  
E. Meerovitch ◽  
E. Ghadirian
Keyword(s):  
Entamoeba Histolytica ◽  
Culture Medium ◽  
Axenic Culture ◽  
The Other ◽  
In Vitro Cultivation ◽  
The Past ◽  
Cholesterol Treatment ◽  
Long Time ◽  
Axenic Conditions

The lost pathogenicity of two strains of Entamoeba histolytica, one isolated in 1924 and the other in 1967, grown in axenic culture for the past 5 and 6 years respectively, was restored by supplementing the culture medium with cholesterol through a number of transfers. The number of passages in the cholesterol-supplemented medium, necessary to restore a certain degree of pathogenicity of the two strains in hamsters, was proportional to the total time of in vitro cultivation of the strain, and not just the time of cultivation under axenic conditions. Pathogenicity, once restored, persisted for a long time after cholesterol treatment was stopped.

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