scholarly journals Preliminary Investigation and Characterization of Electrospun Polycaprolactone and Manuka Honey Scaffolds for Dermal Repair

2015 ◽  
Vol 10 (4) ◽  
pp. 155892501501000 ◽  
Author(s):  
Benjamin A Minden-Birkenmaier ◽  
Rachel M Neuhalfen ◽  
Blythe E Janowiak ◽  
Scott A. Sell
Keyword(s):  
Wound Healing ◽  
Transmission Rate ◽  
Preliminary Investigation ◽  
Manuka Honey ◽  
Gram Negative ◽  
E Coli ◽  
Electrospun Scaffolds ◽  
Group B ◽  
Phosphate Buffered Saline

This study focused on the characterization of Manuka honey-containing poly(ε-caprolactone) (PCL) nanofiber scaffolds with regards to wound healing. Scaffolds were electrospun from 1, 5, 10, and 20% v/v Manuka honey solutions. Scaffolds were subjected to ethanol disinfection and soaked in phosphate-buffered saline (PBS) for various timepoints, and scaffold morphology and honey release was quantified. Scaffolds showed increased water vapor transmission rate (WVTR) with scaffold soak time, indicating an increase in evaporation due to enhanced osmotic potential of the scaffolds. Mechanical testing indicated lower elasticity and strength with honey incorporation, but showed no significant change in material degradation rate with the presence of honey over a 28 day PBS soak. Fibroblast studies showed honey incorporation increased cell infiltration into the scaffold, but scaffold conditioned media did not induce significant chemotaxis towards the scaffold. Honey incorporation also demonstrated an increase in fibroblast proliferation when in direct contact with the scaffolds. Bacterial clearance from pure honey was observed in both Gram positive Streptococcus agalactiae (Group B Streptocococcus) and Gram negative Escherichia coli ( E. coli), but honey scaffolds demonstrated significant clearance in only the Gram negative E. coli. While further investigation is needed, this preliminary study demonstrates the wound-healing potential of Manuka honey-loaded electrospun scaffolds.

Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

2021 ◽  
Author(s):  
Cristina Hernandez Rollan ◽  
Kristoffer Bach Falkenberg ◽  
Maja Rennig ◽  
Andreas Birk Bertelsen ◽  
Morten Norholm
Keyword(s):  
Protein Production ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Family ◽  
Lytic Polysaccharide Monooxygenases ◽  
Strain Bl21 ◽  
Polysaccharide Monooxygenases

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

scholarly journals 480. Molecular Characterization of Multidrug-Resistant Gram-Negative Pathogens in Three Tertiary Care Hospitals in Egypt

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S235-S235
Author(s):  
Amani Kholy ◽  
Samia A Girgis ◽  
Arwa R Elmanakhly ◽  
Mervat A F Shetta ◽  
Dalia El- Kholy ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Resistance Genes ◽  
Genetic Basis ◽  
Tertiary Care ◽  
Carbapenem Resistance ◽  
Resistance Mechanisms ◽  
Gram Negative ◽  
E Coli ◽  
Tertiary Care Hospitals

Abstract Background High rates of AMR among Gram-negative bacilli (GNB) have been reported from Egypt for almost 2 decades. Surveillance and identifying the genetic basis of AMR provide important information to optimize patient care. As there is no adequate data on the genetic basis of AMR in Egypt, we aimed to identify the molecular characterization of multi-drug-resistant (MDR) Gram-negative pathogens (GNP). Methods Three major tertiary-care hospitals in Egypt participated in the “Study for Monitoring Antimicrobial Resistance Trends” (SMART) from 2014 to 2016. Consecutive GNPs were identified and their susceptibility to antimicrobials were tested. Molecular identification of ESBL, AmpC, and carbapenemase resistance genes was conducted on MDR isolates. Results We enrolled 1,070 consecutive Gram-negative isolates; only one isolate per patient according to the standard protocol of (SMART). During 2014–2015, 578 GNP were studied. Enterobacteriaceae comprised 66% of the total isolates. K. pneumoniae and E. coli were the most common (29.8% and 29.4%). K. pneumoniae and E. coli were the predominant organisms in IAI (30.5% and 30.1%, respectively) and UTI (and 38.9% and 48.6%, respectively), while Acinetobacter baumannii was the most prevalent in RTI (40.2%). ESBL producers were phenotypically detected in 53% of K. pneumoniae, and 68% of E. coli. During 2016, 495 GNP were studied. ESBL continued to be high. For E. coli and K. pneunomiea, the most active antimicrobials were amikacin (≥93%), imipenem/meropenem (≥87%) and colistin (97%). Genetic study of ertapenem-resistant isolates and 50% of isolates with ESBL phenotype revealed ESβL production in more than 90% of isolates; blaCTXM-15 was detected in 71.4% and 68.5% in K. pneumoniae and E. coli, respectively, blaTEM-OSBL in 48.5% and47.5% of K. pneumoniae and E. coli, respectively. Carbapenem resistance genes were detected in 45.4% of isolates. In K. pneumoniae, OXA-48 dominated (40.6%), followed by NDM1 (23.7%) and OXA-232 (4.5%). Conclusion Our study detected alarming rates of resistance and identified many resistance mechanisms in clinical isolates from Egyptian hospitals. These high rates highlight the importance of continuous monitoring of the resistance trend and discovering the novel resistant mechanisms of resistance, and the underscores a national antimicrobial stewardship plan in Egypt. Disclosures All authors: No reported disclosures.

scholarly journals Characterization of the β-lactamase specified by the resistance factor R-1818 in Escherichia coli K12 and other Gram-negative bacteria

1971 ◽  
Vol 123 (4) ◽  
pp. 501-505 ◽  
Author(s):  
J. W. Dale
Keyword(s):  
Escherichia Coli ◽  
Host Species ◽  
Host Bacterium ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
R Factor ◽  
Relationship Of ◽  
The Relationship

1. The amino acid composition of the β-lactamase from E. coli (R-1818) was determined. 2. The R-1818 β-lactamase is inhibited by formaldehyde, hydroxylamine, sodium azide, iodoacetamide, iodine and sodium chloride. 3. The Km values for benzylpenicillin, ampicillin and oxacillin have been determined by using the R-factor enzyme from different host species. The same values were obtained, irrespective of the host bacterium. 4. The molecular weight of the enzyme was found to be 44600, and was the same for all host species. 5. The relationship of R-1818 and R-GN238 β-lactamases is discussed.

Synthesis, Characterization, and Antibacterial Evaluation of Curcumin-Sulfanilamide Compound

2020 ◽  
Vol 840 ◽  
pp. 265-269
Author(s):  
Nurjanah Nurjanah ◽  
Endang Saepudin
Keyword(s):  
Antibacterial Activity ◽  
Functional Group ◽  
Biological Activities ◽  
Curcuma Longa ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Ftir Spectrophotometry

Curcumin, a diarylheptanoids compound which isolated primary from Curcuma longa, exhibits a variety of exciting biological activities, including as an antibacterial agent. In the present study, a sulfanilamide-contained curcumin compound was synthesized and characterized to investigate the antibacterial activity against gram-positive bacteria S. aureus, B. subtilis and gram-negative bacteria E. coli. The characterization of the synthesized compound was determined by analysing peak absorbance, functional group, and molecular weight using mass spectroscopy, UV/Vis and FTIR spectrophotometry. Curcumin-sulfanilamide compound exhibited the best antibacterial activity against gram-negative bacteria compared to curcumin and the curcumin-derived compound containing isoxazole with inhibitory zone of 11 mm.

scholarly journals A Comparison of Tissue Engineering Scaffolds Incorporated with Manuka Honey of Varying UMF

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Katherine R. Hixon ◽  
Tracy Lu ◽  
Sarah H. McBride-Gagyi ◽  
Blythe E. Janowiak ◽  
Scott A. Sell
Keyword(s):  
Hydrogen Peroxide ◽  
Tissue Engineering ◽  
Antibacterial Agent ◽  
Antibacterial Properties ◽  
Cell Number ◽  
Bacterial Clearance ◽  
Manuka Honey ◽  
Tissue Engineering Scaffolds ◽  
E Coli ◽  
Electrospun Scaffolds

Purpose. Manuka honey (MH) is an antibacterial agent specific to the islands of New Zealand containing both hydrogen peroxide and a Unique Manuka Factor (UMF). Although the antibacterial properties of MH have been studied, the effect of varying UMF of MH incorporated into tissue engineered scaffolds have not. Therefore, this study was designed to compare silk fibroin cryogels and electrospun scaffolds incorporated with a 5% MH concentration of various UMF.Methods. Characteristics such as porosity, bacterial clearance and adhesion, and cytotoxicity were compared.Results. Pore diameters for all cryogels were between 51 and 60 µm, while electrospun scaffolds were 10 µm. Cryogels of varying UMF displayed clearance of approximately 0.16 cm forE. coliandS. aureus. In comparison, the electrospun scaffolds clearance ranged between 0.5 and 1 cm. A glucose release of 0.5 mg/mL was observed for the first 24 hours by all scaffolds, regardless of UMF. With respect to cytotoxicity, neither scaffold caused the cell number to drop below 20,000.Conclusions. Overall, when comparing the effects of the various UMF within the two scaffolds, no significant differences were observed. This suggests that the fabricated scaffolds in this study displayed similar bacterial effects regardless of the UMF value.

scholarly journals Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

2013 ◽  
Vol 58 (2) ◽  
pp. 722-733 ◽  
Author(s):  
Timothy J. Opperman ◽  
Steven M. Kwasny ◽  
Hong-Suk Kim ◽  
Son T. Nguyen ◽  
Chad Houseweart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Resistance Nodulation Division ◽  
Rnd Efflux Pumps ◽  
Acrab Efflux Pump

ABSTRACTMembers of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC ofEscherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of theEnterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versusE. coliAB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 μM) in combination with 0.016 μg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) ofE. coliAB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 μg/ml CIP alone. In contrast, phenyl-arginine-β-naphthylamide (PAβN), a known EPI, did not increase the bactericidal activity of 0.016 μg/ml CIP at concentrations as high as 100 μM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active againstEnterobacteriaceaespecies andPseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens.

scholarly journals Identification and Biochemical Characterization of a Novel Protein Phosphatase 2C-Like Ser/Thr Phosphatase in Escherichia coli

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Krithika Rajagopalan ◽  
Elizabeth Nagle ◽  
Jonathan Dworkin
Keyword(s):  
Escherichia Coli ◽  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Negative Bacterium ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Novel Protein

Regulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria, including the model Gram-negative bacteriumEscherichia coli, demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thr phosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

scholarly journals Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB

2021 ◽  
Author(s):  
Valérie Biou ◽  
Ricardo Jorge D Adaixo ◽  
Mohamed Chami ◽  
Pierre-Damien Coureux ◽  
Benoist Laurent ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Molecular Motor ◽  
Opportunistic Pathogen ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Class ◽  
Heme Acquisition ◽  
Shed Light

ExbBD is part of a cytoplasmic membrane molecular motor driven by the proton-motive force. It belongs to the larger family of motors involved in nutriment import across the outer membrane of Gram-negative bacteria (ExbBD), flagellar rotation (MotAB) or late steps of cell division in Gram-negative bacteria (TolQR). ExbB and ExbD are integral membrane proteins with three (ExbB) or one (ExbD) transmembrane segment. Here we have solved by single-particle cryo-EM the structures of ExbB alone and of the ExbB-ExbD complex of the opportunistic pathogen Serratia marcescens. ExbBSm alone behaves as a stable pentamer, and the complex displays the ExbB5-ExbD2 stoichiometry. This is similar to what has been observed for ExbB-ExbD complexes from Escherichia coli and Pseudomonas savastanoi as well as MotAB complexes from various species. We identified residues located in the first TM of ExbBSm and ExbBEc that are likely involved in the interaction with TonB/HasB and that are essential for function. ExbBSm has a ca. 40 residues long periplasmic extension absent in E. coli. Such long ExbBs are found in some Gammaproteobacteria, and several genera of Alphaproteobacteria. We show that this extension interacts with HasB, a dedicated TonB paralog from the heme acquisition system (Has) from S. marcescens. We also show that it is involved in heme acquisition via the Has system from S. marcescens. ExbBSm represents thus a new class of ExbB protein and our results shed light on the specificity determinants between the ExbB-ExbD complex and their associated TonB partners.

scholarly journals Detection of Extended-Spectrum β-Lactamases (ESBL) Producing Enterobacteriaceae from Fish Trapped in the Lagoon Area of Bizerte, Tunisia

2020 ◽  
Vol 2020 ◽  
pp. 1-9 ◽  
Author(s):  
Bilel Hassen ◽  
Ahlem Jouini ◽  
Monia Elbour ◽  
Safa Hamrouni ◽  
Abderrazek Maaroufi
Keyword(s):  
Animal Health ◽  
Klebsiella Pneumonia ◽  
Citrobacter Freundii ◽  
Gram Negative ◽  
E Coli ◽  
Shigella Boydii ◽  
Extended Spectrum ◽  
Fish Samples ◽  
Esbl Producers

Extended-spectrum β-lactamase and their molecular mechanism in Enterobacteriaceae were analyzed in 126 fish samples of 9 various wild species, living in the lagoon of Bizerte in Tunisia. Fifty-nine (59) Gram-negative strains were isolated and identified as Escherichia coli (n=24), Klebsiella pneumonia (n=21), Citrobacter freundii (n=8), and Shigella boydii (n=6). Forty-seven ESBL producers were identified using the synergic test. β-Lactamase genes detected were blaCTX-M-1 (E. coli/15; K. pneumonia/8; C. freundii/1; Sh. boydii/1), blaCTX-M-1+ blaOXA-1 (E. coli/4; K. pneumonia/3), blaCTX-M-1+ blaTEM-1-a (K. pneumonia/2), blaCTX-M-15+ blaTEM-1-a (K. pneumonia/1; Sh. boydii/1), blaCTX-M-15+ blaOXA-1 (K. pneumonia/1), blaCTX-M-15 (E. coli/3; K. pneumonia/1; Sh. boydii/3), and blaCTX-M-9 (C. freundii/3). Most strains (84.7%) showed a multiresistant phenotype. qnrA and qnrB genes were identified in six E. coli and in ten E. coli+one K. pneumonia isolates, respectively. The resistance to tetracycline and sulfonamide was conferred by the tet and sul genes. Characterization of phylogenic groups in E. coli isolates revealed phylogroups D (n=20 strains), B2 (n=2), and A (n=2). The studied virulence factor showed prevalence of fimA genes in 9 E. coli isolates (37.5%). Similarly, no strain revealed the three other virulence factors tested (eae, aer, and cnf1). Our findings confirmed that the lagoons of Bizerte may be a reservoir of multidrug resistance/ESBL-producing Enterobacteriaceae. This could lead to indisputable impacts on human and animal health, through the food chain.

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