Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

scholarly journals Applicability of Escherichia Coli As an Indicator for Assessing Quality of Disinfectants and Antiseptic During the COVID-19 Pandemic

2021 ◽  
Author(s):  
Reza Ali Fallahzadeh ◽  
Fariborz Omidi ◽  
Davoud Ghadirian ◽  
Azimeh Fallahzadeh ◽  
Mohammad Reza Nafisi
Keyword(s):  
Test Tube ◽  
Laboratory Method ◽  
Gram Negative Bacteria ◽  
Optimum Conditions ◽  
Gram Negative ◽  
E Coli ◽  
Different Types ◽  
Micro Organisms ◽  
Required Quality

Introduction: The application of disinfectant agents is a common way to fight against micro-organisms. Although there are different types of disinfectant agents to fight COVID-19, many of them do not have the required quality and efficiency. The present work was aimed to evaluate the quality of the available disinfectant agents using gram-negative E. coli bacteria. Materials and Methods: In the laboratory phase of the research, the gram-negative E. coli bacteria were used to evaluate the quality of disinfectants. According to the proposed laboratory method, microbial kit was prepared and used to evaluate the performance of disinfectants. Results: Based on the obtained results, 1.0 mL of microbial suspension in the test tube, as microbial kit, is used for quality assessment of the disinfectants. Also, based on the results and considering the optimum conditions, in case of growth of microorganisms, the quality of disinfectant is evaluated as unfavorable, and in the absence of growth of microorganisms, the quality of disinfectant is considered appropriate in terms of its effect on gram-negative bacteria and weaker microorganisms. Conclusion: Based on the obtained results, E. coli can be used as a reliable indicator for assessing the quality of the disinfectant agents used against COVID 19.

Disinfection of Fruits with Activated Water

2019 ◽  
Vol 10 ◽  
pp. 1864-1872
Author(s):  
Prof. Teodora P. Popova
Keyword(s):  
Antimicrobial Activity ◽  
Aqueous Solutions ◽  
Antimicrobial Properties ◽  
The Other ◽  
Gram Negative Bacteria ◽  
High Antimicrobial Activity ◽  
Gram Negative ◽  
E Coli ◽  
Number Of Microorganisms ◽  
Lower Effect

The effect of ionized aqueous solutions (anolytes and catholyte) in the processing of fruits (cherries, morellos, and strawberries) for decontamination has been tested. Freshly prepared analytes and catholyte without the addition of salts were used, as well as stored for 7 months anolytes, prepared with 0.5% NaCl and a combination of 0.5% NaCl and 0.5% Na2CO3. The anolyte prepared with a combination of 0.5% NaCl and 0.5% Na2CO3, as well as the anolyte obtained with 0.5% NaCl, exhibit high antimicrobial activity against the surface microflora of strawberries, cherries, and sour cherries. They inactivate E. coli for 15 minutes. The other species of the fam. Enterobacteriaceae were also affected to the maximum extent, as is the total number of microorganisms, especially in cherries and sour cherries. Even stored for 7 months, they largely retain their antimicrobial properties. Anolyte and catholyte, obtained without the addition of salts, showed a lower effect on the total number of microorganisms, but had a significant effect on Gram-negative bacteria, and especially with regard to the sanitary indicative E. coli.

Structure-Activity Relationship and Antimicrobial Evaluation of N-Phenylpyrazole Curcumin Derivatives

2020 ◽  
Vol 16 (4) ◽  
pp. 481-488
Author(s):  
Heli Sanghvi ◽  
Satyendra Mishra
Keyword(s):  
Antibacterial Activity ◽  
Gram Negative Bacteria ◽  
Pyrazole Derivatives ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Curcumin Derivatives ◽  
Structure Activity ◽  
Electron Donating Groups ◽  
Derivatives Of

Background: Curcumin, one of the most important pharmacologically significant natural products, has gained significant consideration among scientists for decades since its multipharmacological activities. 1, 3-Dicarbonyl moiety of curcumin was found to be accountable for the rapid degradation of curcumin molecule. The aim of present work is to replace 1, 3-dicarbonyl moiety of curcumin by pyrazole and phenylpyrazole derivatives with a view to improving its stability and to investigate the role of substitution in N-phenylpyrazole curcumin on its antibacterial activity against both Gram-positive as well as Gram-negative bacteria. Methods: Pyrazole derivatives of curcumin were prepared by heating curcumin with phenyhydrazine/ substituted phenyhydrazine derivatives in AcOH. The residue was purified by silica gel column chromatography. Structures of purified compounds were confirmed by 1H NMR and Mass spectroscopy. The synthesized compounds were evaluated for their antibacterial activity by the microdilution broth susceptibility test method against gram positive (S. aureus) and gram negative (E. coli). Results: Effects of substitution in N-phenylpyrazole curcumin derivatives against S. aureus and E. coli were studied. The most active N-(3-Nitrophenylpyrazole) curcumin (12) exhibits twenty-fold more potency against S. aureus (MIC: 10μg/mL)) and N-(2-Fluoroophenylpyrazole) curcumin (5) fivefold more potency against E. coli (MIC; 50 μg/mL) than N-phenylpyrazole curcumin (4). Whereas, a remarkable decline in anti-bacterial activity against S. aureus and E. coli was observed when electron donating groups were incorporated in N-phenylpyrazole curcumin (4). Comparative studies of synthesized compounds suggest the effects of electron withdrawing and electron donating groups on unsubstituted phenylpyrazole curcumin (4). Conclusion: The structure-activity relationship (SAR) results indicated that the electron withdrawing and electron donating at N-phenylpyrazole curcumin played key roles for their bacterial inhibitory effects. The results of the antibacterial evaluation showed that the synthesized pyrazole derivatives of curcumin displayed moderate to very high activity in S. aureus. In conclusion, the series of novel curcumin derivatives were designed, synthesized and tested for their antibacterial activities against S. aureus and E. coli. Among them, N-(3-Nitrophenylpyrazole curcumin; 12) was most active against S. aureus (Gram-positive) and N-(2-Fluoroophenylpyrazole) curcumin (5) against E. coli (Gram-negative) bacteria.

scholarly journals Clonal Clusters, Molecular Resistance Mechanisms and Virulence Factors of Gram-Negative Bacteria Isolated from Chronic Wounds in Ghana

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 339
Author(s):  
Denise Dekker ◽  
Frederik Pankok ◽  
Thorsten Thye ◽  
Stefan Taudien ◽  
Kwabena Oppong ◽  
...  
Keyword(s):  
Molecular Epidemiology ◽  
Virulence Factors ◽  
Iron Uptake ◽  
Chronic Wounds ◽  
Sub Saharan Africa ◽  
Gram Negative Bacteria ◽  
Beta Lactamase ◽  
Secretion Systems ◽  
Gram Negative ◽  
E Coli

Wound infections are common medical problems in sub-Saharan Africa but data on the molecular epidemiology are rare. Within this study we assessed the clonal lineages, resistance genes and virulence factors of Gram-negative bacteria isolated from Ghanaian patients with chronic wounds. From a previous study, 49 Pseudomonas aeruginosa, 21 Klebsiellapneumoniae complex members and 12 Escherichia coli were subjected to whole genome sequencing. Sequence analysis indicated high clonal diversity with only nine P. aeruginosa clusters comprising two strains each and one E. coli cluster comprising three strains with high phylogenetic relationship suggesting nosocomial transmission. Acquired beta-lactamase genes were observed in some isolates next to a broad spectrum of additional genetic resistance determinants. Phenotypical expression of extended-spectrum beta-lactamase activity in the Enterobacterales was associated with blaCTX-M-15 genes, which are frequent in Ghana. Frequently recorded virulence genes comprised genes related to invasion and iron-uptake in E. coli, genes related to adherence, iron-uptake, secretion systems and antiphagocytosis in P. aeruginosa and genes related to adherence, biofilm formation, immune evasion, iron-uptake and secretion systems in K. pneumonia complex. In summary, the study provides a piece in the puzzle of the molecular epidemiology of Gram-negative bacteria in chronic wounds in rural Ghana.

scholarly journals Apomorphine Targets the Pleiotropic Bacterial Regulator Hfq

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 257
Author(s):  
Florian Turbant ◽  
David Partouche ◽  
Omar El Hamoui ◽  
Sylvain Trépout ◽  
Théa Legoubey ◽  
...  
Keyword(s):  
Small Rnas ◽  
Antibacterial Agents ◽  
Amyloid Fibrils ◽  
Noncoding Rnas ◽  
Amyloid Formation ◽  
Gram Negative Bacteria ◽  
Translation Efficiency ◽  
Gram Negative ◽  
Bacterial Adaptation ◽  
E Coli

Hfq is a bacterial regulator with key roles in gene expression. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, thanks to its binding to small regulatory noncoding RNAs. This property is of primary importance for bacterial adaptation and survival in hosts. Small RNAs and Hfq are, for instance, involved in the response to antibiotics. Previous work has shown that the E. coli Hfq C-terminal region (Hfq-CTR) self-assembles into an amyloid structure. It was also demonstrated that the green tea compound EpiGallo Catechin Gallate (EGCG) binds to Hfq-CTR amyloid fibrils and remodels them into nonamyloid structures. Thus, compounds that target the amyloid region of Hfq may be used as antibacterial agents. Here, we show that another compound that inhibits amyloid formation, apomorphine, may also serve as a new antibacterial. Our results provide an alternative in order to repurpose apomorphine, commonly used in the treatment of Parkinson’s disease, as an antibiotic to block bacterial adaptation to treat infections.

scholarly journals Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tessa B. Moyer ◽  
Ashleigh L. Purvis ◽  
Andrew J. Wommack ◽  
Leslie M. Hicks
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Antimicrobial Peptides ◽  
Mechanism Of Action ◽  
Gram Negative Bacteria ◽  
Amaranthus Tricolor ◽  
Core Motif ◽  
Gram Negative ◽  
E Coli ◽  
Membrane Lysis

Abstract Background Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. Results Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. Conclusions This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

scholarly journals An Application of Imipenem Discs or P. aeruginosa ATCC 27853 Reference Strain Increases Sensitivity of Carbapenem Inactivation Method for Non-Fermenting Gram-Negative Bacteria

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 875
Author(s):  
Tomasz Bogiel ◽  
Mateusz Rzepka ◽  
Eugenia Gospodarek-Komkowska
Keyword(s):  
Human Population ◽  
Reference Strain ◽  
Resistance Mechanisms ◽  
Gram Negative Bacteria ◽  
Opportunistic Pathogens ◽  
Beta Lactams ◽  
Gram Negative ◽  
E Coli ◽  
Human Infections ◽  
Reliable Diagnostic Method

Non-fermenting Gram-negative rods are one of the most commonly isolated bacteria from human infections. These microorganisms are typically opportunistic pathogens that pose a serious threat to public health due to possibility of transmission in the human population. Resistance to beta-lactams, due to carbapenemases synthesis, is one of the most important antimicrobial resistance mechanisms amongst them. The aim of this study was to evaluate the usefulness of the Carbapenem Inactivation Method (CIM), and its modifications, for the detection of carbapenemase activity amongst non-fermenting Gram-negative rods. This research involved 81 strains of Gram-negative rods. Of the tested strains, 55 (67.9%) synthesized carbapenemases. For non-fermenting rods, 100% sensitivity and specificity was obtained in the version of the CIM test using imipenem discs and E. coli ATCC 25922 strain. The CIM test allows for differentiation of carbapenems resistance mechanisms resulting from carbapenemase synthesis from other resistance types. It is a reliable diagnostic method for the detection of carbapenemase activity amongst non-fermenting Gram-negative rods. Application of imipenem discs and P. aeruginosa ATCC 27853 reference strain increases CIM results sensitivity, while imipenem discs and E. coli ATCC 25922 strain use maintains full precision of the test for non-fermenting rods.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

2006 ◽  
Vol 50 (7) ◽  
pp. 2478-2486 ◽  
Author(s):  
Andrea Giacometti ◽  
Oscar Cirioni ◽  
Roberto Ghiselli ◽  
Federico Mocchegiani ◽  
Fiorenza Orlando ◽  
...  
Keyword(s):  
Septic Shock ◽  
Antimicrobial Peptide ◽  
Gram Negative Bacteria ◽  
Amphibian Skin ◽  
Necrosis Factor Alpha ◽  
Gram Negative ◽  
Rat Models ◽  
E Coli ◽  
Tnf Α

ABSTRACT Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used β-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and β-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-α levels, resulting in the highest survival rates.

Modular automated bottom-up proteomic sample preparation for high-throughput applications v1

2021 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Tad Ogorzalek ◽  
Paul D. Adams ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
Tryptic Digestion ◽  
Gram Negative Bacteria ◽  
Bottom Up ◽  
Preparation Methods ◽  
Gram Negative ◽  
E Coli ◽  
Protocol Development ◽  
Set Up

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.

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