scholarly journals Dimercaptosuccinic acid in combination with carbapenems against isogenic strains of Escherichia coli producing or not producing a metallo-β-lactamase in vitro and in murine peritonitis

2020 ◽  
Vol 75 (12) ◽  
pp. 3593-3600 ◽  
Author(s):  
G Cheminet ◽  
V de Lastours ◽  
L Poirel ◽  
F Chau ◽  
K Peoc’h ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Peritoneal Fluid ◽  
Dimercaptosuccinic Acid ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bacterial Counts ◽  
E Coli ◽  
Time Kill ◽  
Isogenic Strains

Abstract Background Carbapenemase-producing Enterobacterales represent a major therapeutic challenge. MBLs, requiring zinc at their catalytic site, could be inhibited by meso-dimercaptosuccinic acid (DMSA), a heavy metal chelator already widely used for treating lead intoxication. Objectives To evaluate the activity of carbapenems alone or combined with DMSA against MBL-producing Escherichia coli in a severe murine peritonitis model. Methods Isogenic strains of wild-type E. coli CFT073 producing the MBLs NDM-1, VIM-2 and IMP-1, and the control serine carbapenemases OXA-48 and KPC-3 were constructed. MIC determinations and time–kill assays were performed for imipenem, meropenem and ertapenem alone or in combination with DMSA. Infected mice were treated intraperitoneally for 24 h with imipenem, DMSA or their combination. Bacterial counts in peritoneal fluid and spleen were assessed at 24 h. Results DMSA in combination with each carbapenem caused a significant decrease in the MICs for all MBL-producing strains, in a concentration-dependent manner, but did not provide benefit against non-MBL strains. In mice infected with the NDM-1-producing strain, the combination of imipenem and DMSA significantly reduced bacterial counts in peritoneal fluid (P = 0.0006) and spleen (P < 0.0001), as compared with imipenem alone, with no benefit against the KPC-3-producing and CFT073 strains. DMSA concentrations in plasma of mice were comparable to those obtained in humans with a standard oral dose. Conclusions DMSA restores the activity of carbapenems against MBL-producing strains, and its combination with carbapenems appears to be a promising strategy for the treatment of NDM-producing E. coli infections.

scholarly journals 617. Efficacy of Dimercaptosuccinic Acid (DMSA), a Zinc Chelator, in Combination with Imipenem Against Metallo-β-Lactamase Producing Escherichia coli in a Murine Peritonitis Model

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S287-S287
Author(s):  
Geoffrey Cheminet ◽  
Patrice Nordmann ◽  
Francoise Chau ◽  
Nicolas Kieffer ◽  
Katell Peoc’h ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Maximum Effect ◽  
Dependent Manner ◽  
Bacterial Strains ◽  
Concentration Dependent Manner ◽  
Bacterial Counts ◽  
E Coli ◽  
Zinc Chelator ◽  
Peritonitis Model

Abstract Background A strategy used by bacterial strains to resist β-lactam antibiotics is the expression of metallo-β-lactamases (MBL) requiring zinc for activity. The use of a zinc chelator may restore carbapenem activity against MBL-producing Enterobacteriaceae. DMSA is a heavy metal chelator approved in humans with a satisfactory safety record. Our objective was to evaluate the activity of DMSA in combination with carbapenems, in vitro and in a fatal murine peritonitis model, against MBL-producing Escherichia coli. Methods Isogenic derivatives of wild-type E. coli CFT073 producing the MBL NDM-1, VIM-2, IMP-1, and the serine carbapenemases OXA-48 and KPC-3 were constructed. Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem were determined against each strain alone or in combination with DMSA. Mice were infected with E. coli CFT073 or NDM-1 and treated intraperitoneally for 24 hours with imipenem 100 mg/kg every 4 hours, DMSA 200 mg/kg every 4 hours, or both. Mice survival rates and bacterial counts in peritoneal fluid (PF) and spleen were assessed at 24 hours. Results In vitro, DMSA in combination with each carbapenem permitted a significant decrease of the MICs against all MBL-producing strains, in a concentration-dependent manner. The maximum effect was found for the NDM-1 strain with a 6- to 8-fold MIC reduction, depending on the carbapenem used. NDM-1 strain became susceptible to carbapenems with concentrations of DMSA ≥6 mM. Increasing zinc concentrations above 1 mg/L (average human plasma concentration) did not alter this effect. No benefit of DMSA was observed against non-MBL strains. In vivo, when used alone, the DMSA regimen was not toxic in uninfected mice and ineffective against NDM-1-infected mice (100% mortality). Combination of imipenem and DMSA significantly reduced bacterial counts in PF and spleen as compared with imipenem alone (P < 0.001), and reduced mortality, although not significantly (11% vs. 37%, respectively, P = 0.12). No benefit of the combination was observed against CFT073. Conclusion DMSA is highly effective in vitro in reducing carbapenems MICs against MBL-producing E. coli and appears as a promising strategy in combination with carbapenems for the treatment of NDM-1-related infections. Disclosures All authors: No reported disclosures.

Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity

1992 ◽  
Vol 70 (1) ◽  
pp. 43-48 ◽  
Author(s):  
S. S. Ghosh ◽  
Richard C. Franson
Keyword(s):  
Escherichia Coli ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Phospholipase A ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Streptomyces Chromofuscus ◽  
Hydrolysis Of

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0–8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 μM) or propanolol (IC50 = 180 μM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.Key words: Escherichia coli, substrate, phospholipase D, Streptomyces chromofuscus, sodium fluoride, propranolol.

scholarly journals Cytotoxic Effect of Graphene Oxide Nanoribbons on Escherichia coli

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1339
Author(s):  
Shirong Qiang ◽  
Zhengbin Li ◽  
Li Zhang ◽  
Dongxia Luo ◽  
Rongyue Geng ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Graphene Oxide ◽  
Cytotoxic Effect ◽  
Lactic Dehydrogenase ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Graphene Oxide Nanoribbons ◽  
Graphene Derivatives

The biological and environmental toxicity of graphene and graphene derivatives have attracted great research interest due to their increasing applications. However, the cytotoxic mechanism is poorly understood. Here, we investigated the cytotoxic effect of graphene oxide nanoribbons (GORs) on Escherichia coli (E. coli) in an in vitro method. The fabricated GORs formed long ribbons, 200 nm wide. Based on the results of the MTT assay and plate-culture experiments, GORs significantly inhibited the growth and reproduction of E. coli in a concentration-dependent manner. We found that GORs stimulated E. coli to secrete reactive oxygen species, which then oxidized and damaged the bacterial cell membrane. Moreover, interaction between GORs and E. coli cytomembrane resulted in polysaccharide adsorption by GORs and the release of lactic dehydrogenase. Furthermore, GORs effectively depleted the metal ions as nutrients in the culture medium by adsorption. Notably, mechanical cutting by GORs was not obvious, which is quite different from the case of graphene oxide sheets to E. coli.

Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation

2004 ◽  
Vol 286 (4) ◽  
pp. G645-G652 ◽  
Author(s):  
Keishi Kojima ◽  
Mark W. Musch ◽  
Mark J. Ropeleski ◽  
David L. Boone ◽  
Averil Ma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Map Kinase ◽  
Enteric Bacteria ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Filamentous Actin ◽  
Concentration Dependent Manner ◽  
Epithelial Integrity

Protection of colonic epithelial integrity and function is critical, because compromises in mucosal functions can lead to adverse and potentially life-threatening effects. The gut flora may contribute to this protection, in part, through the sustained induction of cytoprotective heat shock proteins (HSPs) in surface colonocytes. In this study, we investigated whether Escherichia coli LPS mediates bacteria-induced HSP by using cultured young adult mouse colon (YAMC) cells, an in vitro model of the colonic epithelium. E. coli LPS led to an epithelial cell-type specific induction of HSP25 in a time- and concentration-dependent manner, an effect that did not involve changes in HSP72. YAMC cells expressed the toll-like receptors (TLR)2 and TLR4 but not the costimulatory CD14 molecule. Whereas LPS stimulated both the p38 and ERK1/2 but not the stress-activated protein kinase/c-Jun NH2-terminal kinase, signaling pathways in the YAMC cells, all three were stimulated in RAW macrophage cells (in which no LPS-induced HSP25 expression was observed). The p38 inhibitor SB-203580 and the MAP kinase kinase-1 inhibitor PD-98059 inhibited HSP25 induction by LPS. LPS treatment also conferred protection against actin depolymerization induced by the oxidant monochloramine. The HSP25 dependence of the LPS protective effect was outlined in inhibitor studies and through adenovirus-mediated overexpression of HSP25. In conclusion, LPS may be an important mediator of enteric bacteria-induced expression of intestinal epithelial HSP25, an effect that may contribute to filamentous actin stabilization under physiological as well as pathophysiological conditions and thus protection of colonic epithelial integrity.

scholarly journals In Vitro Activity of Trovafloxacin against Bacteroides fragilis in Mixed Culture with either Escherichia coli or a Vancomycin- Resistant Strain of Enterococcus faecium Determined by an Anaerobic Time-Kill Technique

2001 ◽  
Vol 45 (1) ◽  
pp. 243-251 ◽  
Author(s):  
Lorna E. T. Stearne ◽  
Clarissa Kooi ◽  
Wil H. F. Goessens ◽  
Irma A. J. M. Bakker-Woudenberg ◽  
Inge C. Gyssens
Keyword(s):  
Escherichia Coli ◽  
Resistant Strain ◽  
Enterococcus Faecium ◽  
Mixed Cultures ◽  
E Coli ◽  
Abdominal Abscesses ◽  
Pure Cultures ◽  
Vancomycin Resistant ◽  
Time Kill

ABSTRACT To determine the efficacy of trovafloxacin as a possible treatment for intra-abdominal abscesses, we have developed an anaerobic time-kill technique using different inocula to study the in vitro killing ofBacteroides fragilis in pure culture or in mixed culture with either Escherichia coli or a vancomycin-resistant strain of Enterococcus faecium (VREF). With inocula of 5 × 105 CFU/ml and trovafloxacin concentrations of ≤2 μg/ml, a maximum observed effect (E max) of ≥6.1 (log10 CFU/ml) was attained with all pure and mixed cultures within 24 h. With inocula of 108CFU/ml, a similar E max and a similar concentration to produce 50% of E max(EC50) for B. fragilis were found in both pure cultures and mixed cultures with E. coli. However, to produce a similar killing of B. fragilis in the mixed cultures with VREF, a 14-fold increase in the concentration of trovafloxacin was required. A vancomycin-susceptible strain of E. faecium and a trovafloxacin-resistant strain of E. coli were also found to confer a similar “protective” effect on B. fragilis against the activity of trovafloxacin. Using inocula of 109 CFU/ml, the activity of trovafloxacin was retained for E. coli and B. fragilis and was negligible against VREF. We conclude that this is a useful technique to study the anaerobic killing of mixed cultures in vitro and may be of value in predicting the killing of mixed infections in vivo. The importance of using mixed cultures and not pure cultures is clearly shown by the difference in the killing of B. fragilis in the mixed cultures tested. Trovafloxacin will probably be ineffective in the treatment of infections involving large numbers of enterococci. However, due to its ability to retain activity against large cultures of B. fragilis and E. coli, trovafloxacin could be beneficial in the treatment of intra-abdominal abscesses.

scholarly journals Cell-free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs

2021 ◽  
Author(s):  
Hoa Quynh Do ◽  
Carla M Bassil ◽  
Elizabeth I Andersen ◽  
Michaela Jansen
Keyword(s):  
Tumor Cells ◽  
Plasma Membranes ◽  
In Vitro Translation ◽  
Translation System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Membrane Scaffold ◽  
The Impact

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

scholarly journals Antimicrobial Effects of Equine Platelet Lysate

2021 ◽  
Vol 8 ◽  
Author(s):  
Julie Gordon ◽  
Sonsiray Álvarez-Narváez ◽  
John F. Peroni
Keyword(s):  
Bacterial Growth ◽  
Antimicrobial Properties ◽  
Normal Growth ◽  
Platelet Lysate ◽  
Resistant Bacteria ◽  
Growth Media ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli

The development of antimicrobial resistant bacteria and the lack of novel antibiotic strategies to combat those bacteria is an ever-present problem in both veterinary and human medicine. The goal of this study is to evaluate platelet lysate (PL) as a biological alternative antimicrobial product. Platelet lysate is an acellular platelet-derived product rich in growth factors and cytokines that is manufactured via plateletpheresis and pooled from donor horses. In the current study, we sought to define the antimicrobial properties of PL on select gram-positive and gram-negative bacteria. Results from an end-point in vitro assay showed that PL did not support bacterial growth, and in fact significantly reduced bacterial content compared to normal growth media. An in vitro assay was then utilized to further determine the effects on bacterial growth dynamics and showed that all strains exhibited a slower growth rate and lower yield in the presence of PL. The specific effects of PL were unique for each bacterial strain: E. coli and P. aeruginosa growth was affected in a concentration-dependent manner, such that higher amounts of PL had a greater effect, while this was not true for S. aureus or E. faecalis. Furthermore, the onset of exponential growth was delayed for E. coli and P. aeruginosa in the presence of PL, which has significant clinical implications for developing a dosing schedule. In conclusion, our findings demonstrate the potential value of PL as a broad-spectrum antimicrobial that would offer an alternative to traditional antibiotics for the treatment of bacterial infection in equine species.

scholarly journals Epithelial Invasion by Escherichia coli Bearing Dr Fimbriae Is Controlled by Nitric Oxide-Regulated Expression of CD55

2004 ◽  
Vol 72 (5) ◽  
pp. 2907-2914 ◽  
Author(s):  
Li Fang ◽  
Bogdan J. Nowicki ◽  
Petri Urvil ◽  
Pawel Goluszko ◽  
Stella Nowicki ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Epithelial Cell Line ◽  
No Production ◽  
Dependent Manner ◽  
E Coli ◽  
Regulated Expression ◽  
Ishikawa Cells ◽  
Epithelial Invasion

ABSTRACT We previously reported that inhibition of nitric oxide (NO) increases the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr fimbria (Dr+). Epithelial binding and invasion by Dr+ E. coli has also been shown to be dependent upon the expression level of the cellular receptor decay-accelerating factor (DAF; CD55). Here, we hypothesize that NO-related severity of infection could be mediated by changes in DAF expression and in the rate of epithelial invasion. The cellular basis of NO effects on epithelial invasion with Dr+ E. coli was studied using Ishikawa endometrial carcinoma cells as an in vitro model of the human endometrial epithelium. Initially, we show that Ishikawa cells produce NO and express both NO synthase enzymes, NOS II and NOS III, and DAF protein. We next tested the abilities of both Dr+ E. coli and a Dr− E. coli mutant to invade Ishikawa cells, and invasion was seen only with Dr+ E. coli. Invasion by Dr+ E. coli was decreased by elevated NO production and increased by NO inhibition. Elevated NO production significantly decreased DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner. Here, we propose that in vitro invasion of an epithelial cell line is directly related to NO-regulated expression of DAF. The significance of NO-regulated receptor-ligand invasion is that it may represent a novel unrecognized phenomenon of epithelial defense against infection.

Evaluation of Biological Activities of Chemically Synthesized Cobalt Oxide Nanoparticles in Concentration and Time Dependent Manner

2020 ◽  
Vol 13 (6) ◽  
pp. 5243-5249
Author(s):  
Vijayta Gupta ◽  
Vinay Kant ◽  
Meena Sharma
Keyword(s):  
Free Radicals ◽  
Cobalt Oxide ◽  
Biological Activities ◽  
Antibacterial Properties ◽  
Co3o4 Nanoparticles ◽  
Dependent Manner ◽  
Bacterial Strains ◽  
Concentration Dependent Manner ◽  
E Coli

The promising results of metal oxides nanoparticles in different areas including the biological system lead us to investigate the antioxidant and antimicrobial actions of chemically synthesized cobalt oxide (Co3O4) nanoparticles. The different concentrations of synthesized Co3O4 nanoparticles were prepared and evaluated for different parameters at different time intervals i.e.  on day 1, 30 and 60 after preparations.  Co3O4 nanoparticles synthesized in this study were of 52.2 nm average size with a polydispersity index of 0.465. We observed that Co3O4 nanoparticles scavenge different in vitro free radicals (DPPH, ABTS, superoxide anion and hydrogen peroxide radicals) in concentration dependent manner. The percentage of inhibitions of free radicals by Co3O4 nanoparticles was markedly more on day 1 as compared to day 30 and 60. The IC50 values of Co3O4 nanoparticles for these free radicals were also on day 1 as compared to day 30 and 60. The Co3O4 nanoparticles showed the antibacterial actions against both the bacterial strains i.e. S. aureus and E. coli. The MIC and MBC values revealed that action of Co3O4 nanoparticles was more against E. coli than S. aureus. The MIC and MBC values were lower on day 1 as compared to day 30 and 60 with respective to specific bacteria. In conclusions, the Co3O4 nanoparticles synthesized in this study showed potent antioxidant and antibacterial properties due to which it may serve as promising candidate for the combat the biological problems humans, animals and plants associated with reactive oxygen species and bacteria.

scholarly journals Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC

2003 ◽  
Vol 185 (9) ◽  
pp. 2920-2926 ◽  
Author(s):  
Wilson B. Muse ◽  
Christopher J. Rosario ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Assimilation ◽  
Cytosine Deaminase ◽  
In Vitro Transcription ◽  
Dependent Manner ◽  
E Coli ◽  
Different Response ◽  
Control Protein

ABSTRACT Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. β-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position −59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions −120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions −83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

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