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

2014 ◽  
Vol 59 (1) ◽  
pp. 276-281 ◽  
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).

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 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.

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 Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Axel B. Janssen ◽  
Toby L. Bartholomew ◽  
Natalia P. Marciszewska ◽  
Marc J. M. Bonten ◽  
Rob J. L. Willems ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Colistin Resistance ◽  
Anionic Lipid ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Two Component ◽  
Resistant Strains

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

scholarly journals Bacterial Metal Resistance: Coping with Copper without Cooperativity?

mBio ◽  
2021 ◽  
Author(s):  
Nicholas P. Greene ◽  
Vassilis Koronakis
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Cell Envelope ◽  
Metal Resistance ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Rnd Transporter ◽  
Entire Cell

In Escherichia coli and other Gram-negative bacteria, tripartite efflux pumps (TEPs) span the entire cell envelope and serve to remove noxious molecules from the cell. CusBCA is a TEP responsible for copper and silver detoxification in E. coli powered by the resistance-nodulation-cell division (RND) transporter, CusA.

scholarly journals Correct Sorting of Lipoproteins into the Inner and Outer Membranes ofPseudomonas aeruginosaby theEscherichia coliLolCDE Transport System

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Christian Lorenz ◽  
Thomas J. Dougherty ◽  
Stephen Lory
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Transport Systems ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Outer Membranes

ABSTRACTBiogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. InEnterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that inPseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those fromEscherichia coli. TheP. aeruginosalipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either theE. coliorP. aeruginosaLolCDE. We further demonstrate that an inhibitor ofE. coliLolCDE is active againstP. aeruginosaonly when expressing theE. coliorthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCEGram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways ofEscherichia coliandPseudomonas aeruginosaare interchangeable, with theE. coliorthologues correctly sorting theP. aeruginosalipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified inE. colias aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

scholarly journals Fluorescence High-Throughput Screening for Inhibitors of TonB Action

2017 ◽  
Vol 199 (10) ◽  
Author(s):  
Brittany L. Nairn ◽  
Olivia S. Eliasson ◽  
Dallas R. Hyder ◽  
Noah J. Long ◽  
Aritri Majumdar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Compound Libraries

ABSTRACT Gram-negative bacteria acquire ferric siderophores through TonB-dependent outer membrane transporters (TBDT). By fluorescence spectroscopic hgh-throughput screening (FLHTS), we identified inhibitors of TonB-dependent ferric enterobactin (FeEnt) uptake through Escherichia coli FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441 compounds, we evaluated 20 in secondary tests: TonB-dependent ferric siderophore uptake and colicin killing and proton motive force-dependent lactose transport. Six of 20 primary hits inhibited TonB-dependent activity in all tests. Comparison of their effects on [59Fe]Ent and [14C]lactose accumulation suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, are likely not proton ionophores and may inhibit TonB-ExbBD. The facility of FLHTS against E. coli led us to adapt it to Acinetobacter baumannii. We identified its FepA ortholog (AbaFepA), deleted and cloned its structural gene, genetically engineered 8 Cys substitutions in its surface loops, labeled them with fluorescein, and made fluorescence spectroscopic observations of FeEnt uptake in A. baumannii. Several Cys substitutions in AbaFepA (S279C, T562C, and S665C) were readily fluoresceinated and then suitable as sensors of FeEnt transport. As in E. coli, the test monitored TonB-dependent FeEnt uptake by AbaFepA. In microtiter format with A. baumannii, FLHTS produced Z′ factors 0.6 to 0.8. These data validated the FLHTS strategy against even distantly related Gram-negative bacterial pathogens. Overall, it discovered agents that block TonB-dependent transport and showed the potential to find compounds that act against Gram-negative CRE (carbapenem-resistant Enterobacteriaceae)/ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Our results suggest that hundreds of such chemicals may exist in larger compound libraries. IMPORTANCE Antibiotic resistance in Gram-negative bacteria has spurred efforts to find novel compounds against new targets. The CRE/ESKAPE pathogens are resistant bacteria that include Acinetobacter baumannii, a common cause of ventilator-associated pneumonia and sepsis. We performed fluorescence high-throughput screening (FLHTS) against Escherichia coli to find inhibitors of TonB-dependent iron transport, tested them against A. baumannii, and then adapted the FLHTS technology to allow direct screening against A. baumannii. This methodology is expandable to other drug-resistant Gram-negative pathogens. Compounds that block TonB action may interfere with iron acquisition from eukaryotic hosts and thereby constitute bacteriostatic antibiotics that prevent microbial colonization of human and animals. The FLHTS method may identify both species-specific and broad-spectrum agents against Gram-negative bacteria.

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