Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used ...extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3' truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5' truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The aim of the work was to describe the different in vitro models for testing synergism of antibiotics and gather the results of antibiotic synergy against multidrug-resistant Acinetobacter baumannii ...(MDR-Ab). The different original articles were obtained from different web sites. In order to compare the results obtained by the different methods for synergy testing, the Pearson chi-square and the Fischer tests were used. Moreover, non-parametric chi-square test was used in order to compare the frequency distribution in each analysed manuscript. In the current meta-analysis 24 manuscripts, which encompassed 2016 tests of in vitro synergism of different antimicrobials against MDR-Ab, were revised. Checkerboard synergy testing was used in 11 studies, which encompasses 1086 tests (53.9%); time-kill assays were applied in 12 studies, which encompass 359 tests (17.8%); gradient diffusion methods were used in seven studies, encompassing 293 tests (14.5%). And, finally, time-kill plus checkerboard were applied in two studies, encompassing 278 tests (13.8%). By comparing these data, checkerboard and time-kill methods were significantly more used than gradient diffusion methods (p<0.005). Regarding synergy rates obtained on the basis of the applied method, checkerboard provided 227 tests (20.9%) with a synergistic effect; time-kill assays yielded 222 tests (61.8%) with a synergistic effect; gradient diffusion methods only provided 29 tests (9.9%) with a synergistic effect; and, finally, time-kill plus checkerboard yielded just 15 tests (5.4%) with a synergistic effect. When comparing these percentages, synergy rates reported by time-kill methods were significantly higher than that obtained by checkerboard and gradient diffusion methods (p<0.005). On the basis of the revised data, the combinations of a bactericidal antibiotic plus Tigecycline, Vancomycin or Teicoplanin are not recommended. The best combinations of antibiotics are those which include bactericidal antibiotics such as Carbapenems, Fosfomycin, Amikacin, Polymyxins, Rifampicin and Ampicillin/Sulbactam.
•We gather results of antibiotic synergy against Acinetobacter baumannii.•Checkerboard and time-kill methods were more used than gradient diffusion.•Synergy rates by time-kill were higher than that by checkerboard and gradient diffusion.•The best antibiotic combinations are those which include bactericidal antibiotics.
Recently, MALDI-TOF has emerged as a quick tool for bacterial typing. The aim was to evaluate if MALDI-TOF based typing of Legionella pneumophila can achieve the same discriminatory power as that of ...the Sequence Based Typing (SBT) method.
The Sequence Type (ST) was obtained from the 90 strains included in the training set and an in-house MALDI-TOF library based on the Main Spectra Profile (MSP) was generated for the identification of such ST. Then, our library was validated by three procedures: a) creating a dendrogram, b) searching for specific peaks present exclusively in each MSP entry, and c) analysing a validation set composed of 14 strains with known ST. Fully characterized L. pneumophila ATCC 33152, which belongs to ST 36, was used as a control strain. Results: In the training set, 17 strains belonged to ST 1, 1 to ST 20, 63 to ST 22, 1 to ST 146, 6 to ST 578, and 2 to ST 1086. Specific peaks present in each MSPs spectrum, which are considered type-specific biomarkers, ranged from 2 to 11; more concretely, MSP for ST 1 identification shows 2 specific peaks; MSP for ST 20 identification: 9 specific peaks; MSP for ST 22 and ST 36 identification: 11 specific peaks; MSP for ST 146 identification: 5 specific peaks; and MSP for ST 578 and ST 1086 identification: 3 specific peaks. Using the validation set (nine strains belonging to ST 22 and five to ST 1), MALDI-TOF assigned accurately the ST in 30 min per tested strain with a full match.
The ST of L. pneumophila can be identified and reported in few minutes directly from colonies grown on BCYE agar using MALDI-TOF.
•A MALDI-TOF library for the L. pneumophila Sequence Type identification is provided.•Each entry of the library contains its specific biomarker peaks.•L. pneumophila ST can be identified in minutes after colony growing using MALDI-TOF.
Currently, Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) is being evaluated for its efficacy as a fast bacterial typing tool due to its great speed ...compared to other molecular methods. In this study, we evaluated MALDI-TOF as a tool for quick identification and typing of Francisella tularensis.
This study encompassed 86 strains from two different geographical origins (Spain and the Czech Republic), which were previously characterised by Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA). The direct colony method was used for microbial identification. High-quality spectra of the 86 strains were obtained and their main spectra profiles (MSPs) were created for epidemiological typing using MALDI-TOF. Based on the MSPs, principal components were generated and a dendrogram was constructed. An in-house MALDI-TOF library entry was created for each group of PFGE and MLVA strains based on their high-quality spectra. Two dendrograms were obtained using these entries and the unique peaks in each entry were searched.
All strains were correctly identified to the species level. No clear divisions were found in the 86-strain dendrogram; however, Spanish and Czech strains appeared separately in dendrograms created using MLVA and PFGE entries. Entries from our in-house MALDI-TOF library revealed 2–4 biomarker peaks for the detection of the five PFGE groups and 1–12 biomarker peaks for the detection of the seven MLVA groups. Finally, two and one specific biomarkers were found in the Czech and Spanish strains, respectively.
MALDI-TOF can be used to accurately identify F. tularensis strains in less than 15 min. Moreover, data on geographical origin and PFGE and MLVA groups could be obtained in less than one hour after colony growing.
•Mass Spectrometry correctly identifies Francisella tularensis strains in less than 15 min, even if protein extraction is not performed.•A reliable clustering could not be achieved by the dendrogram obtained of all strains.•Dendrograms generated from the entries of our in-house library could be useful for the determination of the geographical origin of F. tularensis strains.
A new arrangement for membrane-assisted liquid-liquid microextraction is presented. The extracting organic solvent was placed into a chromatographic microvial, compatible with the chromatograph ...autosampler, whose septum was replaced by a disc of porous hydrophobic membrane. This extraction device was completely immersed into the analytical sample contained in a cylindrical container subjected to rotary and basculant movement. Then, the extraction of analytes took place from the sample to the organic solvent contained in the vial through the membrane. Esters of the phthalic acid have been selected as model analytes to determine the performance characteristics of the extraction system. The limits of detection, limits of quantification and relative standard deviations (%) were in the range 0.1–0.4, 0.3–1 and 4–7, respectively. Esters of phthalic acid have been successfully analysed in alcoholic beverages. The main operational advantages of this arrangement consisted of minimal required handling, minimal risk of cross contamination and its simplicity.
Aim
To evaluate the serological response against SARS-CoV-2 in a multicenter study representative of the Spanish COVID pandemic.
Methods
IgG and IgM + IgA responses were measured on 1466 samples from ...1236 Spanish COVID-19 patients admitted to the hospital, two commercial ELISA kits (Vircell SL, Spain) based on the detection of antibodies against the viral spike protein and nucleoprotein, were used.
Results
Approximately half of the patients presented antibodies (56.8% were IgM + IgA positive and 43.0% were IgG positive) as soon as 2 days after the first positive PCR result. Serological test positivity increased with time from the PCR test, and 10 days after the first PCR result, 91.5% and 88.0% of the patients presented IgM + IgA and IgG antibodies, respectively.
Conclusion
The high values of sensitivity attained in the present study from a relatively early period of time after hospitalization support the use of the evaluated serological assays as supplementary diagnostic tests for the clinical management of COVID-19.
Matrix‐Assisted Laser Desorption/Ionization‐Time of Flight Mass Spectrometry is a widely used proteomic technique in clinical microbiology laboratories, and enables microbial identification directly ...from clinical samples. This study seeks to establish a protocol for bacterial identification from monomicrobial urine samples that have tested positive in the screening with Sysmex UF‐1000i (Sysmex Corporation, Kobe, Japan). Sysmex UF‐1000i counts ≥1 × 107 bacteria/mL indicate a sufficient bacterial concentration to allow direct identification from urine, with 87.5% sensitivity. Microbial identification from urine with Sysmex UF‐1000i counts between 1 × 105 and 1 × 107 bacteria/ml requires preincubation to obtain the adequate amount of bacteria needed for analysis, and 91.7% sensitivity thus being achieved.
The aim of this work was to ascertain the usefulness of a new commercially-available single-assay chemiluminescence test (CHT) for the diagnosis of human tularemia (Tularaemia VIRCLIA IgG + IgM ...monotest, Vircell, Santa Fe, Granada, Spain). A total of 773 sera from 773 patients including 364 initial sera from patients with diagnosed tularemia, patients with suspected tularemia not confirmed (100), healthy people (152), patients with serology positive to
Brucella
(97), patients diagnosed with other infectious diseases (30), and patients diagnosed with autoimmune diseases (30) were included. All sera were tested by CHT, “in-house” microagglutination test (MAT), immunochromatographic test (ICT) (Virapid Tularaemia, Vircell, Santa Fe Granada, Spain), and “in-house” ELISA IgG, and ELISA IgM. Of the total initial sera, 334 (sensitivity 91.8%) were positive in the CHT, 332 (sensitivity 91.2%) in the MAT, 330 (sensitivity 90.7%) in the ICT, and 328 (sensitivity 90.1%) in the ELISA IgG and ELISA IgM tests. The specificity of the CHT was 96.7%; of the MAT, 100%; of the ICT, 98.7%; and of the ELISA IgG and ELISA IgM, 97.4%. In the group of patients with serology positive to
Brucella
, at least 12.4% of sera were positive in tularemia tests (12.4% in ELISA IgM, 13.4% in MAT, 14.4% in ICT, and 15.5% in CHT and ELISA IgG). In conclusion, CHT presents a sensitivity and specificity in early diagnosis of human tularemia, similar to MAT, ICT, and ELISA IgG and ELISA IgM. Its single assay design allows lower costs, especially in areas of low endemicity or inter-epidemic periods.
Abstract Purpose To perform epidemiological surveillance of Legionella pneumophila in recreational swimming pools in the city of Valladolid (Spain), an area with a continental climate and low ...incidence of legionella-associated infections. Additionally, wild-type minimum inhibitory concentration (MIC) distributions for eight antibiotics commonly used for the treatment of legionellosis were calculated from the isolates obtained. Methods Twelve recreational pools were enrolled between June 2003 and December 2016 and 7221 water samples were taken from three different points of the water network (tank, tap and shower). Legionella culture was performed according to ISO 11731 and 11731-2 standards. MICs of antibiotics were obtained by a gradient test. Results 1.44% of the water samples were positive for L. pneumophila. 60 strains (57.69%) were isolated from showers, 26 (25.00%) from tanks and 18 (17.31%) from taps. L. pneumophila counts were <100 CFU/L in 75 samples (72.12%), 100–1000 CFU/L in 17 (16.35%) and >1000 CFU/L in 12 (11.54%). The MIC90 values obtained were for Rifampicin 0.125 mg/L; Trimethoprim-Sulfamethoxazole 0.25 mg/L; Azithromycin and Levofloxacin 0.5 mg/L; Clarithromycin and Ciprofloxacin 1.0 mg/L; Doxycycline and Tigecycline 4.0 mg/L. Conclusions The use of showers in recreational pools can become a potential pathway for exposure to L. pneumophila , even in cold climates. The wild-type MIC distributions presented in this article may be useful for a better detection of antibiotic resistance and can contribute to improvements in the choice of the antibiotic treatment of legionellosis.
The rapid identification and antibiotic susceptibility test of bacteria causing bloodstream infections are given a very high priority by clinical laboratories. In an effort to reduce the time ...required for performing antibiotic susceptibility test (AST), we have developed a new method to be applied from positive blood culture bottles. The design of method was performed using blood culture bottles prepared artificially with five strains which have a known susceptibility. An aliquot of the blood culture was subcultured in the presence of specific antibiotics and bacterial counts were monitored using the Sysmex UF-1000i flow cytometer at different times up to 180min. Receiver operating curve (ROC) analysis allowed us to find out the cut-off point for differentiating between sensitive and resistant strains to the tested antibiotic. This procedure was then validated against standard commercial methods on a total of 100 positive blood culture bottles from patients. First, bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) directly from positive blood culture bottles as we have previously reported. Secondly, antibiotic susceptibility test was performed in the same way that was carried out in artificially prepared blood culture bottles. Our results indicate that antibiotic susceptibility test can be determined as early as 120min since a blood culture bottle is flagged as positive. The essential agreement between our susceptibility test and commercial methods (E-test, MicroScan and Vitek) was 99%. In summary, we conclude that reliable results on bacterial identification and antibiotic susceptibility test performed directly from positive blood culture bottles can be obtained within 3h.
•A method to determine susceptibility from positive blood cultures is proposed.•The essential agreement between our susceptibility test and commercial methods was 99%.•Results on bacterial identification and susceptibility can be obtained within 3h.
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