Development of a Novel Direct Bioautography-Thin-Layer Chromatography Test: Optimization of Growth Conditions for Gram-Positive Bacteria, Bacillus subtilis

2013 ◽  
Vol 96 (2) ◽  
pp. 386-391 ◽  
Author(s):  
Edyta M Grzelak ◽  
Barbara Majer-Dziedzic ◽  
Irena M Choma ◽  
Karol M Pilorz
Keyword(s):  
Bacillus Subtilis ◽  
Antimicrobial Susceptibility ◽  
Growth Conditions ◽  
Factors Affecting ◽  
Gram Positive Bacteria ◽  
Agar Disc ◽  
Mueller Hinton ◽  
Bromide Solution ◽  
Tlc Plates ◽  
Direct Bioautography

Abstract A TLC-direct bioautography (DB) assay using Bacillus subtilis as test bacteria was developed. Various factors affecting the microorganism's viability on the TLC plates were studied and verified for the flumequine standards. The Dhenasar's method called "direct sample determination" was used for TLC; the antibiotic samples were spotted on the TLC plates and subjected to bioautography without developing with a mobile phase. The best preincubation and incubation times of bacterial broth were found to be 1 h at 37°C and 6 h at 37°C. The optimal viscosity of broth was obtained by the addition of agarose to obtain a 0.05% solution in the Mueller-Hinton broth. The best incubation time of seeded TLC plates was 17 h at 37°C. The plates were visualized by spraying with 0.2% aqueous 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide solution and incubated again for 0.5 h at 37°C. The method was validated by determination of linearity, interday and intraday precision, LOD, and LOQ. The calibration curves showed good linearity in the range 0.005–0.5 μg (0.5–50.0 μg/mL). The regression coefficients were 0.9970 and 0.9955 for intraday and interday plots, respectively. The LOD of flumequine equalled 0.5 μg/mL, i. e., 5 ng of the antibiotic in the spot. The sensitivity of the developed TLC-DB test was compared with that of the two most commonly used standard antimicrobial susceptibility assays: agar disc diffusion and agar cylinder diffusion. The obtained minimum inhibitory concentration values clearly indicate much higher sensitivity of the TLC-DB method compared to the standard antimicrobial susceptibility assays.

Development of a Novel Direct Bioautography–Thin-Layer Chromatography Test: Optimization of Growth Conditions for Gram-Negative Bacteria, Escherichia coli

2011 ◽  
Vol 94 (5) ◽  
pp. 1567-1572 ◽  
Author(s):  
Edyta M Grzelak ◽  
Barbara Majer-Dziedzic ◽  
Irena M Choma
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Intermediate Precision ◽  
Mueller Hinton ◽  
Diphenyltetrazolium Bromide ◽  
Tlc Plates ◽  
Direct Bioautography ◽  
Test Optimization ◽  
Layer Chromatography ◽  
Mueller Hinton Broth

Abstract With the aim of developing a TLC-direct bioautography assay using Escherichia coli as test bacteria, various parameters influencing the viability of microorganisms on TLC plates were examined and checked for flumequine standards. The optimal times for preincubation and incubation of bacterial broth were 20 h at 37°C and 2 h at 37°C, respectively. The optimal viscosity of the broth was obtained for 0.05% agarose solution in Mueller-Hinton broth. Various incubation times of the seeded TLC plates were also tested (5 h proved to be optimal). After incubation, the plates were sprayed with 0.2% aqueous [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide] (MTT) solution and incubated for 0.5 h at 37°C. The precision of the method was evaluated by the repeatability (intraday assay) and intermediate precision (interday assay). The regression coefficients were 0.9977 and 0.9968, respectively, for intraday and interday curves. The calibration curves show good linearity in the range of 0.005–0.50 μg (0.5–50.0 μg/mL). The established LOD of flumequine equaled 0.5 μg/mL, i.e., 5 ng flumequine in the spot. The developed direct bioautography test significantly enhances the sensitivity of the TLC method.

scholarly journals Influence of heterologous MreB proteins on cell morphology of Bacillus subtilis

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3611-3621 ◽  
Author(s):  
Kathrin Schirner ◽  
Jeff Errington
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Shape ◽  
Normal Growth ◽  
Growth Conditions ◽  
Cytoskeletal Protein ◽  
Cell Morphogenesis ◽  
Gram Positive Bacteria ◽  
Related Organism ◽  
Redundant Functions

The prokaryotic cytoskeletal protein MreB is thought to govern cell shape by positioning the cell wall synthetic apparatus at growth sites in the cell. In rod-shaped bacteria it forms helical filaments that run around the periphery of the rod during elongation. Gram-positive bacteria often contain more than one mreB gene. Bacillus subtilis has three mreB-like genes, mreB, mbl and mreBH, the first two of which have been shown to be essential under normal growth conditions. Expression of an mreB homologue from the closely related organism Bacillus licheniformis did not have any effect on cell growth or morphology. In contrast, expression of mreB from the phylogenetically more distant bacterium Clostridium perfringens produced shape defects and ultimately cell death, due to disruption of the endogenous MreB cytoskeleton. However, expression of either mreBB. licheniformis (mreBBl ) or mreBC. perfringens (mreBCp ) was sufficient to confer a rod shape to B. subtilis deleted for the three mreB isologues, supporting the idea that the three proteins have largely redundant functions in cell morphogenesis. Expression of mreBCDBl could fully compensate for the loss of mreBCD in B. subtilis and led to the formation of rod-shaped cells. In contrast, expression of mreBCDCp was not sufficient to confer a rod shape to B. subtilis ΔmreBCD, indicating that a complex of these three cell shape determinants is not enough for cell morphogenesis of B. subtilis.

scholarly journals A high content microscopy screening identifies genes involved in cell width control in Bacillus subtilis

2021 ◽  
Author(s):  
Dimitri Juillot ◽  
Charlene Cornilleau ◽  
Nathalie Deboosere ◽  
Cyrille Billaudeau ◽  
Parfait Evouna-Mengue ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Bacterial Cell ◽  
Growth Conditions ◽  
Cell Diameter ◽  
Genetic Determinants ◽  
Cell Width ◽  
Average Cell ◽  
Gram Positive Bacteria ◽  
State Growth ◽  
Cellular Circuits

How cells control their size is a fundamental question of biology. In bacteria, cell shape is imposed by the extracellular cell wall, in particular by the continuous polymer of peptidoglycan (PG) that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that an error-proof mechanism tightly controls the process. In the rod-shaped model for Gram-positive bacteria, Bacillus subtilis, it is well known that the average cell length varies as a function of growth rate but that cell diameter remains constant throughout its cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width in B. subtilis. Using HCS (high-content screening) fluorescence microscopy and semi-automated measurement of single-cell dimensions, we screened a library of ~ 4000 single knockout mutants. We identified 12 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, these results highlight a link between cell width control and metabolism.

Antibacterial activity of magnesium oxide nanoparticles prepared by Calcination method

2019 ◽  
Vol 10 (03) ◽  
Author(s):  
Elaf Ayad Kadhem ◽  
Miaad Hamzah Zghair ◽  
Sarah , Hussam H. Tizkam, Shoeb Alahmad Salih Mahdi ◽  
Hussam H. Tizkam ◽  
Shoeb Alahmad
Keyword(s):  
Antibacterial Activity ◽  
Magnesium Oxide ◽  
Diffusion Method ◽  
Oxide Nanoparticles ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Magnesium Oxide Nanoparticles ◽  
Agar Disc

magnesium oxide nanoparticles (MgO NPs) were prepared by simple wet chemical method using different calcination temperatures. The prepared NPs were characterized by Electrostatic Discharge (ESD), Scanning Electron Microscope (SEM) and X-ray Diffraction (XRD). It demonstrates sharp intensive peak with the increase of crystallinty and increase of the size with varying morphologies with respect to increase of calcination temperature. Antibacterial studies were done on gram negative bacteria (E.coli) and gram positive bacteria (S.aureus) by agar disc diffusion method. The zones of inhibitions were found larger for gram positive bacteria than gram negative bacteria, this mean, antibacterial MgO NPs activity more active on gram positive bacteria than gram negative bacteria because of the structural differences. It was found that antibacterial activity of MgO NPs was found it has directly proportional with their concentration.

Synthesis of Chalcones and Nucleosides Incorporating [1, 3, 4]Oxadiazolenone Core and Evaluation of their Antifungal and Antibacterial Activities

2020 ◽  
Vol 15 (6) ◽  
pp. 665-679
Author(s):  
Alok K. Srivastava ◽  
Lokesh K. Pandey
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Aspergillus Niger ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Good Antibacterial Activity

Background: [1, 3, 4]oxadiazolenone core containing chalcones and nucleosides were synthesized by Claisen-Schmidt condensation of a variety of benzaldehyde derivatives, obtained from oxidation of substituted 5-(3/6 substituted-4-Methylphenyl)-1, 3, 4-oxadiazole-2(3H)-one and various substituted acetophenone. The resultant chalcones were coupled with penta-O-acetylglucopyranose followed by deacetylation to get [1, 3, 4] oxadiazolenone core containing chalcones and nucleosides. Various analytical techniques viz IR, NMR, LC-MS and elemental analysis were used to confirm the structure of the synthesised compounds.The compounds were targeted against Bacillus subtilis, Staphylococcus aureus and Escherichia coli for antibacterial activity and Aspergillus flavus, Aspergillus niger and Fusarium oxysporum for antifungal activity. Methods: A mixture of Acid hydrazides (3.0 mmol) and N, Nʹ- carbonyl diimidazole (3.3 mmol) in 15 mL of dioxane was refluxed to afford substituted [1, 3, 4]-oxadiazole-2(3H)-one. The resulted [1, 3, 4]- oxadiazole-2(3H)-one (1.42 mmol) was oxidized with Chromyl chloride (1.5 mL) in 20 mL of carbon tetra chloride and condensed with acetophenones (1.42 mmol) to get chalcones 4. The equimolar ratio of obtained chalcones 4 and β -D-1,2,3,4,6- penta-O-acetylglucopyranose in presence of iodine was refluxed to get nucleosides 5. The [1, 3, 4] oxadiazolenone core containing chalcones 4 and nucleosides 5 were tested to determined minimum inhibitory concentration (MIC) value with the experimental procedure of Benson using disc-diffusion method. All compounds were tested at concentration of 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.62 mg/mL, 0.31 mg/mL and 0.15 mg/mL for antifungal activity against three strains of pathogenic fungi Aspergillus flavus (A. flavus), Aspergillus niger (A. niger) and Fusarium oxysporum (F. oxysporum) and for antibacterial activity against Gram-negative bacterium: Escherichia coli (E. coli), and two Gram-positive bacteria: Staphylococcus aureus (S. aureus) and Bacillus subtilis(B. subtilis). Result: The chalcones 4 and nucleosides 5 were screened for antibacterial activity against E. coli, S. aureus and B. subtilis whereas antifungal activity against A. flavus, A. niger and F. oxysporum. Compounds 4a-t showed good antibacterial activity whereas compounds 5a-t containing glucose moiety showed better activity against fungi. The glucose moiety of compounds 5 helps to enter into the cell wall of fungi and control the cell growth. Conclusion: Chalcones 4 and nucleosides 5 incorporating [1, 3, 4] oxadiazolenone core were synthesized and characterized by various spectral techniques and elemental analysis. These compounds were evaluated for their antifungal activity against three fungi; viz. A. flavus, A. niger and F. oxysporum. In addition to this, synthesized compounds were evaluated for their antibacterial activity against gram negative bacteria E. Coli and gram positive bacteria S. aureus, B. subtilis. Compounds 4a-t showed good antibacterial activity whereas 5a-t showed better activity against fungi.

scholarly journals Silver Nanoparticles and Their Antibacterial Applications

2021 ◽  
Vol 22 (13) ◽  
pp. 7202
Author(s):  
Tamara Bruna ◽  
Francisca Maldonado-Bravo ◽  
Paul Jara ◽  
Nelson Caro
Keyword(s):  
Silver Nanoparticles ◽  
Antibacterial Agents ◽  
Multidrug Resistant ◽  
Secondary Effects ◽  
Cytotoxic Effects ◽  
Factors Affecting ◽  
Gram Positive Bacteria ◽  
Resistant Strains

Silver nanoparticles (AgNPs) have been imposed as an excellent antimicrobial agent being able to combat bacteria in vitro and in vivo causing infections. The antibacterial capacity of AgNPs covers Gram-negative and Gram-positive bacteria, including multidrug resistant strains. AgNPs exhibit multiple and simultaneous mechanisms of action and in combination with antibacterial agents as organic compounds or antibiotics it has shown synergistic effect against pathogens bacteria such as Escherichia coli and Staphylococcus aureus. The characteristics of silver nanoparticles make them suitable for their application in medical and healthcare products where they may treat infections or prevent them efficiently. With the urgent need for new efficient antibacterial agents, this review aims to establish factors affecting antibacterial and cytotoxic effects of silver nanoparticles, as well as to expose the advantages of using AgNPs as new antibacterial agents in combination with antibiotic, which will reduce the dosage needed and prevent secondary effects associated to both.

scholarly journals Elucidation of Gram-Positive Bacterial Iron(III) Reduction for Kaolinite Clay Refinement

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3084
Author(s):  
Hao Jing ◽  
Zhao Liu ◽  
Seng How Kuan ◽  
Sylvia Chieng ◽  
Chun Loong Ho
Keyword(s):  
Iron Reduction ◽  
Growth Conditions ◽  
Cellular Interaction ◽  
Kaolin Clay ◽  
Media Composition ◽  
Gram Positive ◽  
Kaolinite Clay ◽  
Gram Positive Bacteria ◽  
Rich Media ◽  
The Media

Recently, microbial-based iron reduction has been considered as a viable alternative to typical chemical-based treatments. The iron reduction is an important process in kaolin refining, where iron-bearing impurities in kaolin clay affects the whiteness, refractory properties, and its commercial value. In recent years, Gram-negative bacteria has been in the center stage of iron reduction research, whereas little is known about the potential use of Gram-positive bacteria to refine kaolin clay. In this study, we investigated the ferric reducing capabilities of five microbes by manipulating the microbial growth conditions. Out of the five, we discovered that Bacillus cereus and Staphylococcus aureus outperformed the other microbes under nitrogen-rich media. Through the biochemical changes and the microbial behavior, we mapped the hypothetical pathway leading to the iron reduction cellular properties, and found that the iron reduction properties of these Gram-positive bacteria rely heavily on the media composition. The media composition results in increased basification of the media that is a prerequisite for the cellular reduction of ferric ions. Further, these changes impact the formation of biofilm, suggesting that the cellular interaction for the iron(III)oxide reduction is not solely reliant on the formation of biofilms. This article reveals the potential development of Gram-positive microbes in facilitating the microbial-based removal of metal contaminants from clays or ores. Further studies to elucidate the corresponding pathways would be crucial for the further development of the field.

scholarly journals In Vitro Characterization of the Bacillus subtilis Protein Tyrosine Phosphatase YwqE

2005 ◽  
Vol 187 (10) ◽  
pp. 3384-3390 ◽  
Author(s):  
Ivan Mijakovic ◽  
Lucia Musumeci ◽  
Lutz Tautz ◽  
Dina Petranovic ◽  
Robert A. Edwards ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Class Ii ◽  
Gram Positive ◽  
Protein Tyrosine ◽  
Gram Positive Bacteria ◽  
New Family ◽  
In Vitro Characterization

ABSTRACT Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains one such CpsB-like PTP, YwqE, in addition to two class II Cys-based PTPs, YwlE and YfkJ. The substrates for both YwlE and YfkJ are presently unknown, while YwqE was shown to dephosphorylate two phosphotyrosine-containing proteins implicated in UDP-glucuronate biosynthesis, YwqD and YwqF. In this study, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE.

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