Phospholipid distribution in the cytoplasmic membrane of Gram-negative bacteria is highly asymmetric, dynamic, and cell shape-dependent

The distribution of phospholipids across the inner membrane (IM) of Gram-negative bacteria is unknown. We demonstrate that the IMs of Escherichia coli and Yersinia pseudotuberculosis are asymmetric, with a 75%/25% (cytoplasmic/periplasmic leaflet) distribution of phosphatidylethanolamine (PE) in rod-shaped cells and an opposite distribution in E. coli filamentous cells. In initially filamentous PE-lacking E. coli cells, nascent PE appears first in the periplasmic leaflet. As the total PE content increases from nearly zero to 75%, cells progressively adopt a rod shape and PE appears in the cytoplasmic leaflet of the IM. The redistribution of PE influences the distribution of the other lipids between the leaflets. This correlates with the tendency of PE and cardiolipin to regulate antagonistically lipid order of the bilayer. The results suggest that PE asymmetry is metabolically controlled to balance temporally the net rates of synthesis and translocation, satisfy envelope growth capacity, and adjust bilayer chemical and physical properties.

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Synergistic Interactions of Vancomycin with Different Antibiotics against Escherichia coli: Trimethoprim and Nitrofurantoin Display Strong Synergies with Vancomycin against Wild-Type E. coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03502-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 276-281 ◽

Cited By ~ 38

Author(s):

Alice Zhou ◽

Tina Manzhu Kang ◽

Jessica Yuan ◽

Casey Beppler ◽

Caroline Nguyen ◽

...

Keyword(s):

Escherichia Coli ◽

The Other ◽

Gram Negative Bacteria ◽

Functional Categories ◽

Wild Type ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Synergistic Interactions ◽

Pairwise Interactions

ABSTRACTGram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We usedEscherichia colimutants that are partially sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-typeE. coliat concentrations of VAN as low as 6.25 μg/ml. This suggests that a very small percentage of exogenous VAN does enterE. colibut normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489–494, 2006,http://dx.doi.org/10.1038/ng1755).

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Disinfection of Fruits with Activated Water

JOURNAL OF ADVANCES IN AGRICULTURE ◽

10.24297/jaa.v10i0.8462 ◽

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.

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

BMC Microbiology ◽

10.1186/s12866-021-02176-4 ◽

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00537-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5995-6002 ◽

Cited By ~ 7

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.

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Interaction of Gram-Negative Bacteria with the Lysosomal Fraction of Polymorphonuclear Leukocytes II. Changes in the Cell Envelope of Escherichia coli

Infection and Immunity ◽

10.1128/iai.1.3.311-318.1970 ◽

1970 ◽

Vol 1 (3) ◽

pp. 311-318

Author(s):

D. Friedberg ◽

I. Friedberg ◽

M. Shilo

Keyword(s):

Escherichia Coli ◽

Polymorphonuclear Leukocytes ◽

Cytoplasmic Membrane ◽

Morphological Changes ◽

Cell Envelope ◽

Gram Negative Bacteria ◽

Intact Cells ◽

Lysosomal Fraction ◽

E Coli ◽

Absorbing Material

Interaction of lysosomal fraction with Escherichia coli caused damage to the cell envelope of these intact cells and to the cytoplasmic membrane of E. coli spheroplasts. The damage to the cytoplasmic membrane was manifested in the release of 260-nm absorbing material and β-galactosidase from the spheroplasts, and by increased permeability of cryptic cells to O -nitrophenyl-β- d -galactopyranoside; damage to the cell wall was measured by release of alkaline phosphatase. Microscope observation showed morphological changes in the cell envelope.

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

Biochemical Journal ◽

10.1042/bj1230501 ◽

1971 ◽

Vol 123 (4) ◽

pp. 501-505 ◽

Cited By ~ 13

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.

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Heterogeneous and Flexible Transmission ofmcr-1in Hospital-AssociatedEscherichia coli

mBio ◽

10.1128/mbio.00943-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 23

Author(s):

Yingbo Shen ◽

Zuowei Wu ◽

Yang Wang ◽

Rong Zhang ◽

Hong-Wei Zhou ◽

...

Keyword(s):

Escherichia Coli ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Genes Encoding

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

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In Vitro Activity of Newer and Conventional Antimicrobial Agents, Including Fosfomycin and Colistin, against Selected Gram-Negative Bacilli in Kuwait

Pathogens ◽

10.3390/pathogens7030075 ◽

2018 ◽

Vol 7 (3) ◽

pp. 75 ◽

Cited By ~ 5

Author(s):

Wadha Alfouzan ◽

Rita Dhar ◽

David Nicolau

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

The Other ◽

In Vitro Activity ◽

Limited Data ◽

Gram Negative ◽

E Coli ◽

Microbroth Dilution

Limited data are available on susceptibilities of these organisms to some of the recently made accessible antimicrobial agents. The in vitro activities of newer antibiotics, such as, ceftolozane/tazobactam (C/T) and ceftazidime/avibactam (CZA) along with some “older” antibiotics, for example fosfomycin (FOS) and colistin (CL) were determined against selected strains (resistant to ≥ 3 antimicrobial agents) of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Minimum inhibitory concentrations (MIC) were determined by Clinical and Laboratory Standards Institute microbroth dilution. 133 isolates: 46 E. coli, 39 K. pneumoniae, and 48 P. aeruginosa were tested. Results showed that E. coli isolates with MIC50/90, 0.5/1 μ g / mL for CL; 4/32 μ g / mL for FOS; 0.25/32 μ g / mL for C/T; 0.25/8 μ g / mL for CZA, exhibited susceptibility rates of 95.7%, 97.8%, 76.1%, and 89.1%, respectively. On the other hand, K. pneumoniae strains with MIC50/90, 0.5/1 μ g / mL for CL; 256/512 μ g / mL for FOS; 2/128 μ g / mL for C/T; 0.5/128 μ g / mL for CZA showed susceptibility rates of 92.3%, 7.7%, 51.3%, and 64.1%, respectively. P. aeruginosa isolates with MIC50/90, 1/1 μ g / mL for CL; 128/128 μ g / mL for C/T; 32/64 μ g / mL for CZA presented susceptibility rates of 97.9%, 33.3%, and 39.6%, respectively. Higher MICs were demonstrated against most of the antibiotics. However, CL retained efficacy at low MICs against most of the isolates tested.

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National Surveillance of Antimicrobial Susceptibility of Bacteremic Gram-Negative Bacteria with Emphasis on Community-Acquired Resistant Isolates: Report from the 2019 Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01089-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Po-Yu Liu ◽

Yu-Lin Lee ◽

Min-Chi Lu ◽

Pei-Lan Shao ◽

Po-Liang Lu ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Gram Negative Bacteria ◽

National Surveillance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Carbapenem Resistant

ABSTRACT A multicenter collection of bacteremic isolates of Escherichia coli (n = 423), Klebsiella pneumoniae (n = 372), Pseudomonas aeruginosa (n = 300), and Acinetobacter baumannii complex (n = 199) was analyzed for susceptibility. Xpert Carba-R assay and sequencing for mcr genes were performed for carbapenem- or colistin-resistant isolates. Nineteen (67.8%) carbapenem-resistant K. pneumoniae (n = 28) and one (20%) carbapenem-resistant E. coli (n = 5) isolate harbored blaKPC (n = 17), blaOXA-48 (n = 2), and blaVIM (n = 1) genes.

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Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

The Journal of Lipid Research ◽

10.1194/jlr.ra120000654 ◽

2020 ◽

Vol 61 (6) ◽

pp. 870-883 ◽

Cited By ~ 2

Author(s):

Inga Nilsson ◽

Sheng Y. Lee ◽

William S. Sawyer ◽

Christopher M. Baxter Rath ◽

Guillaume Lapointe ◽

...

Keyword(s):

Escherichia Coli ◽

Gram Negative Bacteria ◽

Metabolic Labeling ◽

Wild Type ◽

Transport Pathways ◽

Gram Negative ◽

E Coli ◽

Outer Membranes ◽

Outer Leaflet ◽

Promising Tool

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.

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