scholarly journals Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

10.3791/62903 ◽  
2021 ◽  
Author(s):  
Josselin Rigaill ◽  
Estelle Audoux ◽  
Killian Rodriguez ◽  
Aurélien Peyron ◽  
Philippe Berthelot ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Compounds ◽  
Protection Assay ◽  
Enzyme Protection

scholarly journals Highlighting the Biotechnological Potential of Deep Oceanic Crust Fungi through the Prism of Their Antimicrobial Activity

Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 411
Author(s):  
Maxence Quemener ◽  
Marie Dayras ◽  
Nicolas Frotté ◽  
Stella Debaets ◽  
Christophe Le Meur ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Oceanic Crust ◽  
Gene Clusters ◽  
Multidrug Resistant ◽  
Antimicrobial Activities ◽  
Antimicrobial Compounds ◽  
Human Pathogens ◽  
Biotechnological Potential ◽  
Fungal Isolates

Among the different tools to address the antibiotic resistance crisis, bioprospecting in complex uncharted habitats to detect novel microorganisms putatively producing original antimicrobial compounds can definitely increase the current therapeutic arsenal of antibiotics. Fungi from numerous habitats have been widely screened for their ability to express specific biosynthetic gene clusters (BGCs) involved in the synthesis of antimicrobial compounds. Here, a collection of unique 75 deep oceanic crust fungi was screened to evaluate their biotechnological potential through the prism of their antimicrobial activity using a polyphasic approach. After a first genetic screening to detect specific BGCs, a second step consisted of an antimicrobial screening that tested the most promising isolates against 11 microbial targets. Here, 12 fungal isolates showed at least one antibacterial and/or antifungal activity (static or lytic) against human pathogens. This analysis also revealed that Staphylococcus aureus ATCC 25923 and Enterococcus faecalis CIP A 186 were the most impacted, followed by Pseudomonas aeruginosa ATCC 27853. A specific focus on three fungal isolates allowed us to detect interesting activity of crude extracts against multidrug-resistant Staphylococcus aureus. Finally, complementary mass spectrometry (MS)-based molecular networking analyses were performed to putatively assign the fungal metabolites and raise hypotheses to link them to the observed antimicrobial activities.

scholarly journals Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Jin-Hahn Kim ◽  
Akhilesh Kumar Chaurasia ◽  
Nayab Batool ◽  
Kwan Soo Ko ◽  
Kyeong Kyu Kim
Keyword(s):  
Host Cells ◽  
Intracellular Bacteria ◽  
Protection Assay ◽  
Host Cell Membrane ◽  
Bacterial Killing ◽  
Content Type ◽  
Gentamicin Protection Assay ◽  
Enzyme Protection ◽  
The Difference ◽  
Kinetics Of

ABSTRACTPrecise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellularStaphylococcus aureus. We found that the difference between the two methods for the recovery of intracellular CFU ofS. aureuswas about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

Identification and Semiquantitative Estimation of Antibiotics Added to Complete Feeds, Premixes, and Concentrates

1999 ◽  
Vol 82 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Jean-Louis Gafner
Keyword(s):  
Staphylococcus Aureus ◽  
Quality Assurance ◽  
Uv Light ◽  
Acid Extraction ◽  
Antimicrobial Compounds ◽  
Routine Method ◽  
Agar Diffusion ◽  
Microbiological Methods ◽  
Layer Chromatography ◽  
Feed Samples

Abstract Classical microbiological methods for determining antimicrobial compounds in feeds are nonspecific. Thus, there is a need to identify biological activity, and bioautography is used for thispurpose. A routine method for detecting the following antimicrobial sub stances in feeds is described: avilamycin, avoparcin, Zn-bacitracin, erythromycin, flavomycin, furazolidone, lasalocid, monensin, narasin, penicillin, salinomycin, spiramycin, tetracyclines, tylosin, and virginiamycin. Carbadox can be detected by UV light examination of the plates prior to bioautography. Semiquantitative estimations of antibiotic content are compared with quantitative determinations of the above mentioned sub stances in feeds, except erythromycin, penicillin, and tetracyclines. Detection limits range from 0.1 mg/kg (chlortetracycline) to 20 mg/kg (lasalocid). The method involves agar diffusion of buffered samples, a neutral extraction of polyether antibiotics followed bythin-layer chromatography (TLC), and an acid extraction for other antibiotics followed by TLC. Five test bacteria were used for the main detection by agar diffusion: Micrococcus luteusATCC 9341, Staphylococcus aureus ATCC 6538P, Corynebacterium xerosis NCTC 9755, Bacillus cereus ATCC 11778, and B. subtilis ATCC 6633. Identification after TLC was achieved by bioautography with the most sensitive microorganism(s). This method allows one laboratory technician to analyze up to 30 feed samples within 2.5 working days, provided that feeds of the same category are analyzed in the same run, and that labels of additives are available. Qualitative and semiquantitative information are valuable when performing a quantitative antibiotic determination and it provides proof that the activity determined is due to the tested substance. This last feature is essential from the perspective of quality assurance of results.

scholarly journals Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

2004 ◽  
Vol 383 (3) ◽  
pp. 551-559 ◽  
Author(s):  
Sheraz GUL ◽  
Richard BROWN ◽  
Earl MAY ◽  
Marie MAZZULLA ◽  
Martin G. SMYTH ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dna Ligase ◽  
Protection Assay ◽  
Dna Ligases ◽  
Enzyme Substrate ◽  
Ic50 Values ◽  
Acridinium Ester

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

scholarly journals MIKROBA ENDOFIT DARI TANAMAN SRIKAYA (Annona squamosa L.) SEBAGAI PENGHASIL ANTIMIKROBA Staphylococcus aureus DAN Candida albicans

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Ruth Melliawati ◽  
Sunifah Sunifah
Keyword(s):  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
High Performance ◽  
Agar Medium ◽  
Agar Plate ◽  
Antimicrobial Compounds ◽  
Cyanogenic Glycosides ◽  
Annona Squamosa ◽  
Chloroform Phase ◽  
Apple Plant

Various studies indicated that endophytic microbes lived in the plant tissues and produced antimicrobial compounds. Sugar-apple plant  (Annona squamosa L) contained alkaloids, cyanogenic glycosides, and flavonoids. The purpose of this reasearch were (1) to determine the endophytic microbes isolated from sugar-apple plant (2) to study inhibiting capabillity of endophytic isolate against Staphylococcus aureus and Candida albicans, (3) to analyze antimicrobial compounds produced by the potential endophytic isolate. Diffusion agar plate methode was used to assessed antimicrobial activity. Antimicrobial compounds were analyzed by Thin Layer Chormatography (TLC) and High Performance Liquid Chormatography (HPLC), compared with erythromycin, metronidazole and tetracycline. Twelve bacterial isolates and 24 fungus were isolated. Selected bacteria, BMC 1.1, showed the biggest clear zone on C. albicans culture on agar medium, meanwhile selected fungi, BTCK 1.1T, formed the biggest colony on S. aureus culture on agar medium. TLC and HPLC analysis showed that the Rf value of BMC 1.1 and BTCK 1.1T chloroform phase fractions was similiar to metronidazole. Metronidazole concentration in C1, C2, Ck1 and Ck2 fraction were 170.98 ppm, 18.27 ppm, 1.51 ppm and 4.14 ppm respectively.

scholarly journals Optimization of Medium Fermentation for Production of Antimicrobial Compounds by Endofit Bacteria Andalas Plant (Morus macroura Miq.) B.J.T.A-6 Isolate

BioScience ◽  
2019 ◽  
Vol 3 (1) ◽  
pp. 79
Author(s):  
Nada Nafion ◽  
Dwi Hilda Putri ◽  
Irdawati Irdawati
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Endophytic Bacteria ◽  
Bacterial Resistance ◽  
Inhibition Zone ◽  
Fermentation Medium ◽  
Luria Bertani ◽  
Antimicrobial Compounds ◽  
Luria Bertani Broth ◽  
The World

Cases of bacterial resistance to antibiotics are discussed with serious problems in the world of health. New antimicrobial compounds are needed which are more effective in treating infectious diseases. Isolate B.J.T.A-6 is an endophytic bacteria from Andalas plants (Morus macroura Miq.) which is known to be able to produce antimicrobial active compounds. Antimicrobial compounds can be produced by growing them on fermented media. The purpose of this study was to optimize the medium fermentation of Andalas endophytic bacteria of B.J.T.A-6  isolates in producing antimicrobial compounds. While medium fermentation is Nutrient Broth (NB), Muller Hinton (MH), and Luria Bertani Broth (LB). Antimicrobial activity tests were carried out by means of diffusion. The parameters used were the diameter of the inhibition zone against Staphylococcus aureus around the disc paper. The profile of medium fermentation optimization was analyzed statistically. The results showed that B.J.T.A-6  isolates could inhibit the highest growth of S. aureus by using LB fermentation medium. 

Bacteriocin Production by Lactic Acid Bacteria Isolated from Regional Cheeses: Inhibition of Foodborne Pathogens

1994 ◽  
Vol 57 (11) ◽  
pp. 1013-1015 ◽  
Author(s):  
MARIA E. FARIAS ◽  
AIDA A. P. DE RUIZ HOLGADO ◽  
FERNANDO SESMA
Keyword(s):  
Heat Treatment ◽  
Staphylococcus Aureus ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Foodborne Pathogens ◽  
Bacteriocin Production ◽  
Antimicrobial Compounds ◽  
Heat Stable ◽  
And Storage ◽  
And Staphylococcus Aureus

Four strains of enterococci isolated from Argentina regional cheeses were found to produce bacteriocins that were active against several lactic acid bacteria. Among them, enterocin CRL35 produced by Enterococcus faecium CRL35 was also inhibitory to foodborne pathogens like Listeria monocytogenes and Staphylococcus aureus. These antimicrobial compounds were sensitive to proteases and heat stable; inhibitory activity of enterocin CRL35 showed also to be stable at extreme pHs, heat treatment, and storage in different conditions.

scholarly journals Cold-adapted bacterial extracts as a source of anti-infective and antimicrobial compounds against Staphylococcus aureus

2019 ◽  
Vol 14 (16) ◽  
pp. 1369-1382 ◽  
Author(s):  
Marco Artini ◽  
Rosanna Papa ◽  
Gianluca Vrenna ◽  
Concetta Lauro ◽  
Annarita Ricciardelli ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Marine Bacteria ◽  
Biofilm Formation ◽  
Medical Applications ◽  
Polar Regions ◽  
Antimicrobial Compounds ◽  
Cold Adapted ◽  
Organic Extracts

Aim: The dramatic emergence of antibiotic resistance has directed the interest of research toward the discovery of novel antimicrobial molecules. In this context, cold-adapted marine bacteria living in polar regions represent an untapped reservoir of biodiversity endowed with an interesting chemical repertoire. The aim of this work was to identify new antimicrobials and/or antibiofilm molecules produced by cold-adapted bacteria. Materials & methods: Organic extracts obtained from polar marine bacteria were tested against Staphylococcus aureus. Most promising samples were subjected to suitable purification strategies. Results: Results obtained led to the identification of a novel lipopeptide able to effectively inhibit the biofilm formation of S. aureus. Conclusion: New lipopeptide may be potentially useful in a wide variety of biotechnological and medical applications.

Intracellular activity of antimicrobial compounds used for Staphylococcus aureus nasal decolonization

2018 ◽  
Vol 73 (11) ◽  
pp. 3044-3048 ◽  
Author(s):  
J Rigaill ◽  
M F Morgene ◽  
M Gavid ◽  
Y Lelonge ◽  
Z He ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Compounds ◽  
Intracellular Activity
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