scholarly journals Effect of Lactamase Inhibitors on the Biosensor Penp during the Measurement of Lactam Antibiotics Concentration

Sensors ◽  
2019 ◽  
Vol 19 (5) ◽  
pp. 1237
Author(s):  
Dagoberto Soto ◽  
Camila Silva ◽  
Cristian Ugalde ◽  
Kwok-Yin Wong ◽  
Yun-Chung Leung ◽  
...  
Keyword(s):  
Resistance Mechanism ◽  
Common Element ◽  
Clinical Samples ◽  
Fluorescent Biosensor ◽  
Biological Recognition ◽  
Common Strategy ◽  
Lactam Ring ◽  
Mechanism Of Interaction ◽  
Fluorescence Level ◽  
Covalently Linked

PenP is a fluorescent biosensor of lactam antibiotics (LA). It is structurally derived from the mutant lactamase TEM-1 comprising the substitution E166C, where fluorescein is covalently linked to cysteine. The presence of LA in the medium produces a change in the intrinsic fluorescence level of the biosensor, and the integral of the fluorescence level over time correlates directly with the LA concentration. Previously, we have successfully used PenP to determine the concentration of lactam antibiotics in clinical samples. The use of lactamase inhibitors (LI) is a common strategy to enhance the effect of LA due to the inhibition of an important resistance mechanism of pathogenic microorganisms. Structurally, LI and LA share the common element of recognition of lactamases (the lactam ring), but they differ in the reversibility of the mechanism of interaction with said enzyme. Because the biological recognition domain of PenP is derived from a lactamase, LI is expected to interfere with the PenP detection capabilities. Surprisingly, this work provides evidence that the effect of LI is marginal in the determination of LA concentration mediated by PenP.

scholarly journals Mutation-Based Antibiotic Resistance Mechanism in Methicillin-Resistant Staphylococcus aureus Clinical Isolates

2021 ◽  
Vol 14 (5) ◽  
pp. 420
Author(s):  
Tanveer Ali ◽  
Abdul Basit ◽  
Asad Mustafa Karim ◽  
Jung-Hun Lee ◽  
Jeong-Ho Jeon ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Active Site ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Meca Gene ◽  
Resistance Mechanism ◽  
Ligand Docking ◽  
Clinical Samples ◽  
Allosteric Site ◽  
Methicillin Resistant

β-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against β-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards β-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.

scholarly journals PREVALENCE OF CEPHALOSPORIN-RESISTANT GRAM-NEGATIVE BACILLI FROM CLINICAL SAMPLES

2016 ◽  
pp. 176
Author(s):  
Kavi Aniis ◽  
Rajamanikandan Kcp ◽  
Arvind Prasanth D
Keyword(s):  
Clinical Samples ◽  
Beta Lactamase ◽  
Bacterial Isolates ◽  
Beta Lactam ◽  
Gram Negative ◽  
Beta Lactamases ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Lactam Ring ◽  
Esbl Producers

<p>ABSTRACT<br />Objective: Beta-lactams are the group of antibiotics that contain a ring called as “beta-lactam ring,” which is responsible for the antibacterial activity.<br />The presence of resistance among Gram-negative organisms is due to the production of beta-lactamases enzymes that hydrolysis the beta-lactam ring<br />thereby conferring resistance to the organism. This study is undertaken to determine the prevalence of extended-spectrum beta-lactamase (ESBL)<br />producing Gram-negative organism from clinical samples.<br />Methods: A total of 112 clinical samples were taken for this study. The combined disc synergistic test (CDST) was used for the phenotypic detection<br />of ESBL producers from the clinical samples. The genotypic identification of ESBL producers was carried out by alkaline lysis method by isolation of<br />plasmid DNA.<br />Result: A total of 87 bacterial isolates were isolated and identified. Among them, Klebsiella (41%) was the predominant organism followed by<br />Escherichia coli (33%), Proteus (10%), Pseudomonas (10%), and Serratia (6%). Among the various bacterial isolates, Klebsiella showed a higher<br />percentage of resistance. The CDST showed that 8 isolates of Klebsiella, 3 isolates of E. coli, and 1 isolate of Pseudomonas were found to be ESBL<br />producers. The genotypic confirmation showed that the two bacterial isolates, namely, Klebsiella and E. coli were found to possess temoniera (TEM)<br />gene which was the 400-500 bp conferring resistance to the antibiotics.<br />Conclusion: The results of this study suggest that early detection of ESBL producing Gram-negative organism is a very important step in planning the<br />therapy of patient in Hospitals. CDST continues to be a good indicator in the detection of ESBL producers.<br />Keywords: Beta-lactamases, Gram-negative bacilli, Extended-spectrum beta-lactamase, Resistance, Combined disc synergistic test.</p><p> </p>

Column affinity chromatography for bound/free separation in ligand assays. I. Radioimmunoassay of choriomammotropin (human placental lactogen).

1981 ◽  
Vol 27 (6) ◽  
pp. 896-900 ◽  
Author(s):  
P Cornale ◽  
M Bonazzi ◽  
C Multinu ◽  
P Romelli ◽  
L Vancheri ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Solid Phase ◽  
Placental Lactogen ◽  
Assay Method ◽  
Clinical Samples ◽  
Good Precision ◽  
Phase Reaction ◽  
Pass Through ◽  
Covalently Linked ◽  
Polyethylene Glycol Precipitation

Abstract A method is described for separating antibody-bound from free fractions in ligand assays by column affinity chromatography, and its application to radioimmunoassay of choriomammotropin. In the method, 70 x 10 mm (i.d.) polypropylene columns containing about 150 mg of immunosorbent (goat anti-rabbit gamma-globulins covalently linked to Sepharose CL-4B) are used. Standards or unknowns, tracer and antiserum, pipetted into bottom-capped columns, are kept separated from the immunosorbent bed by a porous polyethylene disc and allowed to react for 15 min at room temperature. The reaction mixture is then allowed to pass through the columns by removing the bottom caps. Free antigen is eluted by washing the column, and discarded; antibody-bound fractions remain bound to the immunosorbent. The radioactivity in the columns is counted. The major advantages of the present technique, arising from the liquid-phase reaction combined with the solid-phase separation by column affinity chromatography, are the very low nonspecific binding (less than 1%), good sensitivity (0.02 mg/L), good precision (CV 3.4%), and simple and fast (30-min) assay. For 50 clinical samples so assayed (gamma) and compared with a polyethylene glycol precipitation technique (x), the regression equation was: y - 0.14 + 0.98x (r = 0.994). The assay method was clinical validated by 3493 determinations.

BacARscan: A Comprehensive and Interactive Web-Resource to Discern Antibiotic Resistance Gene Diversity in –Omics Datasets

2021 ◽  
Author(s):  
Deeksha Pandey ◽  
Bandana Kumari ◽  
Neelja Singhal ◽  
Manish Kumar
Keyword(s):  
Antibiotic Resistance ◽  
Gene Diversity ◽  
Antibiotic Resistance Gene ◽  
Resistance Mechanism ◽  
Antibiotic Resistance Genes ◽  
Metagenomic Data ◽  
Clinical Samples ◽  
Antibiotics Resistance ◽  
Web Resource ◽  
Proteomic Data

Abstract Regular surveillance of antibiotic resistance genes (ARGs) is important to understand the emergence and epidemiology of antibiotic resistance (AR) in clinical and environmental niches. With diminishing costs, NGS technologies are anticipated to replace classical microbiological and molecular methods for determination of AR. One major hindrance underlying identification and annotation of ARGs from WGS data is that a major part of genome databases contain fragmented genes/genomes (due to incomplete assembly). Herein, we propose a web resource of Bacterial ARGs, named as BacARscan (Bacterial Antibiotic Resistance scan), to detect, predict and characterize ARGs in metagenomic, genomic and proteomic data. The current version of BacARscan comprises 254 ARG models, each annotated with a resistant profile against different classes of antibiotics, resistance mechanism etc. Benchmarking on a combined dataset of AR and non-AR proteins found 92% precision & 95% F-measure. BacARscan can also discriminate between the protein families that are homologous but not all families are involved in the AR. BacARscan identified more ARGs in (a) gut microbiome and (b) datasets comprising short read genomic and proteomic sequences of ESKAPE pathogens. Analysis of clinical metagenomic data indicated its potential to complement and/or supplement WGS based identification of ARGs in clinical samples. BacARscan standalone software and web-server are freely available at http://proteininformatics.org/mkumar/bacarscan and github repository (https://github.com/University-of-Delhi-south-campus/BacARscan).

scholarly journals Innovative DendrisChips® Technology for a Syndromic Approach of In Vitro Diagnosis: Application to the Respiratory Infectious Diseases

Diagnostics ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 77 ◽  
Author(s):  
Alice Senescau ◽  
Tatiana Kempowsky ◽  
Elodie Bernard ◽  
Sylvain Messier ◽  
Philippe Besse ◽  
...  
Keyword(s):  
Laser Scanner ◽  
Pcr Amplification ◽  
Clinical Samples ◽  
Decision Algorithm ◽  
Detection And Identification ◽  
Glass Slides ◽  
Syndromic Approach ◽  
Covalently Linked ◽  
Species Specific

Clinical microbiology is experiencing the emergence of the syndromic approach of diagnosis. This paradigm shift will require innovative technologies to detect rapidly, and in a single sample, multiple pathogens associated with an infectious disease. Here, we report on a multiplex technology based on DNA-microarray that allows detecting and discriminating 11 bacteria implicated in respiratory tract infection. The process requires a PCR amplification of bacterial 16S rDNA, a 30 min hybridization step on species-specific oligoprobes covalently linked on dendrimers coated glass slides (DendriChips®) and a reading of the slides by a dedicated laser scanner. A diagnostic result is delivered in about 4 h as a predictive value of presence/absence of pathogens using a decision algorithm based on machine-learning method, which was constructed from hybridization profiles of known bacterial and clinical isolated samples and which can be regularly enriched with hybridization profiles from clinical samples. We demonstrated that our technology converged in more than 95% of cases with the microbiological culture for bacteria detection and identification.

scholarly journals cyp51A-Based Mechanisms of Aspergillus fumigatus Azole Drug Resistance Present in Clinical Samples from Germany

2013 ◽  
Vol 57 (8) ◽  
pp. 3513-3517 ◽  
Author(s):  
Oliver Bader ◽  
Michael Weig ◽  
Utz Reichard ◽  
Raimond Lugert ◽  
Martin Kuhns ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistance Mechanism ◽  
Cross Resistance ◽  
Clinical Samples ◽  
Steady Increase ◽  
Clinical Test ◽  
Content Type ◽  
Visible Growth ◽  
Resistant Strains ◽  
High Level

ABSTRACTSince the mid-1990s, a steady increase in the occurrence of itraconazole-resistantAspergillus fumigatusisolates has been observed in clinical contexts, leading to therapeutic failure in the treatment of aspergillosis. This increase has been predominantly linked to a single allele of thecyp51Agene, termed TR/L98H, which is thought to have arisen through the use of agricultural azoles. Here, we investigated the current epidemiology of triazole-resistantA. fumigatusand underlyingcyp51Amutations in clinical samples in Germany. From a total of 527 samples, 17 (3.2%) showed elevated MIC0values (the lowest concentrations with no visible growth) for at least one of the three substances (itraconazole, voriconazole, and posaconazole) tested. The highest prevalence of resistant isolates was observed in cystic fibrosis patients (5.2%). Among resistant isolates, the TR/L98H mutation incyp51Awas the most prevalent, but isolates with the G54W and M220I substitutions and the novel F219C substitution were also found. The isolate with the G54W substitution was highly resistant to both itraconazole and posaconazole, while all others showed high-level resistance only to itraconazole. For the remaining six isolates, no mutations incyp51Awere found, indicating the presence of other mechanisms. With the exception of the strains carrying the F219C and M220I substitutions, many itraconazole-resistant strains also showed cross-resistance to voriconazole and posaconazole with moderately increased MIC0values. In conclusion, the prevalence of azole-resistantA. fumigatusin our clinical test set is lower than that previously reported for other countries. Although the TR/L98H mutation frequently occurs among triazole-resistant strains in Germany, it is not the only resistance mechanism present.

Detection of viruses by electron microscopy: Increased sensitivity by direct micro-ultracentrifugation of samples

1987 ◽  
Vol 45 ◽  
pp. 908-909
Author(s):  
Alain R. Trudel ◽  
M. Trudel
Keyword(s):  
Electron Microscopy ◽  
Fold Increase ◽  
Observation Time ◽  
Clinical Samples ◽  
Maximum Speed ◽  
Viral Particles ◽  
Structural Details ◽  
Latex Spheres ◽  
Increased Sensitivity ◽  
Size Of Particles

AirfugeR (Beckman) direct ultracentrifugation of viral samples on electron microscopy grids offers a rapid way to concentrate viral particles or subunits and facilitate their detection and study. Using the A-100 fixed angle rotor (30°) with a K factor of 19 at maximum speed (95 000 rpm), samples up to 240 μl can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10 to 40 fold increase in sensitivity depending on the size of particles. This technique also permits quantification of viral particles in samples if an aliquot is mixed with latex spheres of known concentration.Direct ultracentrifugation for electron microscopy can be performed on laboratory samples such as gradient or column fractions, infected cell supernatant, or on clinical samples such as urine, tears, cephalo-rachidian liquid, etc..

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

1994 ◽  
Vol 52 ◽  
pp. 276-277
Author(s):  
Mary Beth Downs ◽  
Wilson Ribot ◽  
Joseph W. Farchaus
Keyword(s):  
Cell Wall ◽  
Molecular Sieves ◽  
Cell Envelope ◽  
Single Species ◽  
Supporting Cell ◽  
Surface Layers ◽  
Rabbit Igg ◽  
In Vitro Assembly ◽  
Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

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