scholarly journals Proton motive force underpins respiration-mediated potentiation of aminoglycoside lethality in pathogenic Escherichia coli

2022 ◽  
Vol 204 (1) ◽  
Author(s):  
Calum M. Webster ◽  
Ayrianna M. Woody ◽  
Safura Fusseini ◽  
Louis G. Holmes ◽  
Gary K. Robinson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Respiratory Chain ◽  
Proton Motive Force ◽  
Motive Force ◽  
Gram Negative Bacteria ◽  
Ros Production ◽  
Gram Negative ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Aminoglycoside Toxicity

AbstractIt is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli. DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides.

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 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 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.

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

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
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.

scholarly journals 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)

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.

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

2020 ◽  
Vol 61 (6) ◽  
pp. 870-883 ◽  
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.­

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

2020 ◽  
Vol 6 (23) ◽  
pp. eaaz6333 ◽  
Author(s):  
Mikhail Bogdanov ◽  
Kyrylo Pyrshev ◽  
Semen Yesylevskyy ◽  
Sergey Ryabichko ◽  
Vitalii Boiko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Physical Properties ◽  
Cell Shape ◽  
Yersinia Pseudotuberculosis ◽  
Cytoplasmic Membrane ◽  
The Other ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Phospholipid Distribution

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.

Escherichia coli tol and rcs genes participate in the complex network affecting curli synthesis

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2487-2497 ◽  
Author(s):  
Anne Vianney ◽  
Grégory Jubelin ◽  
Sophie Renault ◽  
Corine Dorel ◽  
Philippe Lejeune ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Membrane Integrity ◽  
Regulatory Proteins ◽  
Gram Negative Bacteria ◽  
Dependent Manner ◽  
Dominant Effect ◽  
Gram Negative ◽  
E Coli

Curli are necessary for the adherence of Escherichia coli to surfaces, and to each other, during biofilm formation, and the csgBA and csgDEFG operons are both required for their synthesis. A recent survey of gene expression in Pseudomonas aeruginosa biofilms has identified tolA as a gene activated in biofilms. The tol genes play a fundamental role in maintaining the outer-membrane integrity of Gram-negative bacteria. RcsC, the sensor of the RcsBCD phosphorelay, is involved, together with RcsA, in colanic acid capsule synthesis, and also modulates the expression of tolQRA and csgDEFG. In addition, the RcsBCD phosphorelay is activated in tol mutants or when Tol proteins are overexpressed. These results led the authors to investigate the role of the tol genes in biofilm formation in laboratory and clinical isolates of E. coli. It was shown that the adherence of cells was lowered in the tol mutants. This could be the result of a drastic decrease in the expression of the csgBA operon, even though the expression of csgDEFG was slightly increased under such conditions. It was also shown that the Rcs system negatively controls the expression of the two csg operons in an RcsA-dependent manner. In the tol mutants, activation of csgDEFG occurred via OmpR and was dominant upon repression by RcsB and RcsA, while these two regulatory proteins repressed csgBA through a dominant effect on the activator protein CsgD, thus affecting curli synthesis. The results demonstrate that the Rcs system, previously known to control the synthesis of the capsule and the flagella, is an additional component involved in the regulation of curli. Furthermore, it is shown that the defect in cell motility observed in the tol mutants depends on RcsB and RcsA.

scholarly journals Identification of Two New Proteins in Spermidine Nucleoids Isolated from Escherichia coli

1999 ◽  
Vol 181 (12) ◽  
pp. 3842-3844 ◽  
Author(s):  
Lizabeth D. Murphy ◽  
Judah L. Rosner ◽  
Steven B. Zimmerman ◽  
Dominic Esposito
Keyword(s):  
Escherichia Coli ◽  
Functional Form ◽  
Dnase I ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Form Analysis ◽  
E Coli ◽  
Amino Terminal

ABSTRACT The Escherichia coli nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type E. coli. Partial amino-terminal sequencing has identified them as the products of rdgC andyejK. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.

scholarly journals Occurrence of Escherichia coli in captive psittaciformes: antimicrobial susceptibility and virulence genes

2020 ◽  
Vol 21 ◽  
Author(s):  
Karine Louise Calaça ◽  
Renato Clini Cervi ◽  
Silvânia Andrade Reis ◽  
Iolanda Aparecida Nunes ◽  
Valéria de Sá Jayme ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Susceptibility ◽  
Virulence Genes ◽  
Susceptibility Testing ◽  
Metropolitan Region ◽  
Serum Resistance ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Hygienic Conditions

Abstract Captive Psittaciformes may harbor Gram-negative bacteria in their digestive tract, mainly due to poor hygienic conditions and confinement. The present study was carried out with the objective of isolating and identifying Escherichia coli in samples collected from Psittaciformes cages in 50 commercial establishments in the metropolitan region of Goiania, with subsequent antimicrobial susceptibility testing and detection of virulence genes. A total of 141 samples of excreta and swab samples from feeders and water bowls were collected, totaling 423 samples. Escherichia coli was isolated from 9.7% (41/423) samples: 12% (17/141) in excreta, 8.5% (12/141) in feed, and 8.5% (12 /141) in waterers. To determine the susceptibility profile of E. coli isolates, resistance to ciprofloxacin 4.9% (2/41), gentamicin 17.0% (7/41), doxycycline 34.1% (14/41), florfenicol 34.1% (14/41), trimethoprim 39.0% (16/41), tetracycline 41.5% (17/41), enrofloxacin 43.9% (18/41), amoxicillin 48.8% (20/41), neomycin 61.0% (25/41), and sulfonamide 90.2% (37/41) was determined. In 20 isolates, resistance was determined at 4 or more antimicrobials, seven of excreta (7/17), five of feed (5/12), and eight of waterers (8/12). One of the isolates from the waterers showed resistance to all antimicrobials. The iss gene was detected in three isolates, the tsh gene in three, the papC gene in two, traT and eae genes were not detected. In this study, it can be concluded that Psittaciformes commercialized as pet are carry E. coli isolates resistant to most commonly used antimicrobials, mainly sulfonamides and neomycin, besides having virulence and serum resistance genes, which highlights the possibility of the to cause disease in humans.

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