scholarly journals Recovery of Plasmid pEMB1, Whose Toxin-Antitoxin System Stabilizes an Ampicillin Resistance-Conferring β-Lactamase Gene in Escherichia coli, from Natural Environments

2014 ◽  
Vol 81 (1) ◽  
pp. 40-47 ◽  
Author(s):  
Hyo Jung Lee ◽  
Hyun Mi Jin ◽  
Moon Su Park ◽  
Woojun Park ◽  
Eugene L. Madsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Stability ◽  
Functional Characterization ◽  
Treatment Plant ◽  
Antibiotic Resistance Genes ◽  
Cloning And Expression ◽  
Natural Environments ◽  
Ampicillin Resistance ◽  
Content Type ◽  
Lactamase Gene

ABSTRACTNon-culture-based procedures were used to investigate plasmids showing ampicillin resistance properties in two different environments: remote mountain soil (Mt. Jeombong) and sludge (Tancheon wastewater treatment plant). Total DNA extracted from the environmental samples was directly transformed intoEscherichia coliTOP10, and a single and three different plasmids were obtained from the mountain soil and sludge samples, respectively. Interestingly, the restriction fragment length polymorphism pattern of the plasmid from the mountain soil sample, designated pEMB1, was identical to the pattern of one of the three plasmids from the sludge sample. Complete DNA sequencing of plasmid pEMB1 (8,744 bp) showed the presence of six open reading frames, including a β-lactamase gene. Using BLASTX, theorf5andorf6genes were suggested to encode a CopG family transcriptional regulator and a plasmid stabilization system, respectively. Functional characterization of these genes using a knockoutorf5plasmid (pEMB1ΔparD) and the cloning and expression oforf6(pET21bparE) indicated that these genes were antitoxin (parD) and toxin (parE) genes. Plasmid stability tests using pEMB1 and pEMB1ΔparDEinE. colirevealed that theorf5andorf6genes enhanced plasmid maintenance in the absence of ampicillin. Using a PCR-based survey, pEMB1-like plasmids were additionally detected in samples from other human-impacted sites (sludge samples) and two other remote mountain soil samples, suggesting that plasmids harboring a β-lactamase gene with a ParD-ParE toxin-antitoxin system occurs broadly in the environment. This study extends knowledge about the dissemination and persistence of antibiotic resistance genes in naturally occurring microbial populations.

scholarly journals Molecular Epidemiology of NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Isolates from Pakistan

2014 ◽  
Vol 58 (9) ◽  
pp. 5589-5593 ◽  
Author(s):  
Anna L. Sartor ◽  
Muhammad W. Raza ◽  
Shahid A. Abbasi ◽  
Kathryn M. Day ◽  
John D. Perry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Epidemiology ◽  
Genetic Relatedness ◽  
Antibiotic Resistance Genes ◽  
Enterobacter Cloacae ◽  
Content Type ◽  
Plasmid Replicon ◽  
Genes Encoding ◽  
Clonal Spread ◽  
Encoding Genes

ABSTRACTThe molecular epidemiology of 66 NDM-producing isolates from 2 Pakistani hospitals was investigated, with their genetic relatedness determined using repetitive sequence-based PCR (Rep-PCR). PCR-based replicon typing and screening for antibiotic resistance genes encoding carbapenemases, other β-lactamases, and 16S methylases were also performed. Rep-PCR suggested a clonal spread ofEnterobacter cloacaeandEscherichia coli. A number of plasmid replicon types were identified, with the incompatibility A/C group (IncA/C) being the most common (78%). 16S methylase-encoding genes were coharbored in 81% of NDM-producingEnterobacteriaceae.

scholarly journals Novel Conjugative Plasmid from Escherichia coli of Swine Origin That Coharbors the Multiresistance Genecfrand the Extended-Spectrum-β-Lactamase GeneblaCTX-M-14b

2014 ◽  
Vol 59 (2) ◽  
pp. 1337-1340 ◽  
Author(s):  
Wan-Jiang Zhang ◽  
Xiu-Mei Wang ◽  
Lei Dai ◽  
Xin Hua ◽  
Zhimin Dong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conjugative Plasmid ◽  
Content Type ◽  
Conjugative Plasmids ◽  
Extended Spectrum ◽  
Lactamase Gene

ABSTRACTTwo porcineEscherichia coliisolates harbored thecfrgene on conjugative plasmids of 38,405 bp (pGXEC6) and 41,646 bp (pGXEC3). In these two plasmids, thecfrgene was located within a 4,612-bp region containing atnpA-IS26-cfr-IS26-Δhypelement. Plasmid pGXEC3 was almost identical to pGXEC6 except for a 3,235-bp ISEcp1-blaCTX-M-14binsertion. The colocation of the multiresistancecfrgene with an extended-spectrum-β-lactamase gene on a conjugative plasmid may support the dissemination of these genes by coselection.

scholarly journals Complete Circular Genome Sequence of a Multidrug-Resistant Escherichia coli Strain from Cuba Obtained with Nanopore and Illumina Hybrid Assembly

2019 ◽  
Vol 8 (48) ◽  
Author(s):  
Rosa Elena Hernández-Fillor ◽  
Michael Brilhante ◽  
Ivette Espinosa ◽  
Vincent Perreten
Keyword(s):  
Escherichia Coli ◽  
Genome Sequence ◽  
Antibiotic Resistance Genes ◽  
Generation Cephalosporin ◽  
Multidrug Resistant ◽  
Coli Strain ◽  
Hybrid Assembly ◽  
Content Type ◽  
Long Reads ◽  
Cephalosporin Resistance

The complete genome sequence of a multidrug-resistant Escherichia coli strain isolated from a healthy pig in Cuba was determined using short and long reads. This strain carried four plasmids, including a 42,683-kb IncX1 plasmid, which contains the third-generation cephalosporin resistance gene bla CTX-M-32 together with other disinfectant and antibiotic resistance genes.

scholarly journals During Oxidative Stress the Clp Proteins of Escherichia coli Ensure that Iron Pools Remain Sufficient To Reactivate Oxidized Metalloenzymes

2020 ◽  
Vol 202 (18) ◽  
Author(s):  
Ananya Sen ◽  
Yidan Zhou ◽  
James A. Imlay
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Acid Synthesis ◽  
Growth Defect ◽  
Natural Environments ◽  
Intracellular Iron ◽  
Amino Acid Synthesis ◽  
Common Component ◽  
Content Type ◽  
Iron Pool

ABSTRACT Hydrogen peroxide (H2O2) is formed in natural environments by both biotic and abiotic processes. It easily enters the cytoplasms of microorganisms, where it can disrupt growth by inactivating iron-dependent enzymes. It also reacts with the intracellular iron pool, generating hydroxyl radicals that can lethally damage DNA. Therefore, virtually all bacteria possess H2O2-responsive transcription factors that control defensive regulons. These typically include catalases and peroxidases that scavenge H2O2. Another common component is the miniferritin Dps, which sequesters loose iron and thereby suppresses hydroxyl-radical formation. In this study, we determined that Escherichia coli also induces the ClpS and ClpA proteins of the ClpSAP protease complex. Mutants that lack this protease, plus its partner, ClpXP protease, cannot grow when H2O2 levels rise. The growth defect was traced to the inactivity of dehydratases in the pathway of branched-chain amino acid synthesis. These enzymes rely on a solvent-exposed [4Fe-4S] cluster that H2O2 degrades. In a typical cell the cluster is continuously repaired, but in the clpSA clpX mutant the repair process is defective. We determined that this disability is due to an excessively small iron pool, apparently due to the oversequestration of iron by Dps. Dps was previously identified as a substrate of both the ClpSAP and ClpXP proteases, and in their absence its levels are unusually high. The implication is that the stress response to H2O2 has evolved to strike a careful balance, diminishing iron pools enough to protect the DNA but keeping them substantial enough that critical iron-dependent enzymes can be repaired. IMPORTANCE Hydrogen peroxide mediates the toxicity of phagocytes, lactic acid bacteria, redox-cycling antibiotics, and photochemistry. The underlying mechanisms all involve its reaction with iron atoms, whether in enzymes or on the surface of DNA. Accordingly, when bacteria perceive toxic H2O2, they activate defensive tactics that are focused on iron metabolism. In this study, we identify a conundrum: DNA is best protected by the removal of iron from the cytoplasm, but this action impairs the ability of the cell to reactivate its iron-dependent enzymes. The actions of the Clp proteins appear to hedge against the oversequestration of iron by the miniferritin Dps. This buffering effect is important, because E. coli seeks not just to survive H2O2 but to grow in its presence.

scholarly journals F Plasmids Are the Major Carriers of Antibiotic Resistance Genes in Human-Associated Commensal Escherichia coli

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Craig Stephens ◽  
Tyler Arismendi ◽  
Megan Wright ◽  
Austin Hartman ◽  
Andres Gonzalez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Transposable Elements ◽  
Dna Sequencing ◽  
Resistance Genes ◽  
Bacterial Pathogens ◽  
Acquired Resistance ◽  
Antibiotic Resistance Genes ◽  
Content Type ◽  
E Coli

ABSTRACT The evolution and propagation of antibiotic resistance by bacterial pathogens are significant threats to global public health. Contemporary DNA sequencing tools were applied here to gain insight into carriage of antibiotic resistance genes in Escherichia coli, a ubiquitous commensal bacterium in the gut microbiome in humans and many animals, and a common pathogen. Draft genome sequences generated for a collection of 101 E. coli strains isolated from healthy undergraduate students showed that horizontally acquired antibiotic resistance genes accounted for most resistance phenotypes, the primary exception being resistance to quinolones due to chromosomal mutations. A subset of 29 diverse isolates carrying acquired resistance genes and 21 control isolates lacking such genes were further subjected to long-read DNA sequencing to enable complete or nearly complete genome assembly. Acquired resistance genes primarily resided on F plasmids (101/153 [67%]), with smaller numbers on chromosomes (30/153 [20%]), IncI complex plasmids (15/153 [10%]), and small mobilizable plasmids (5/153 [3%]). Nearly all resistance genes were found in the context of known transposable elements. Very few structurally conserved plasmids with antibiotic resistance genes were identified, with the exception of an ∼90-kb F plasmid in sequence type 1193 (ST1193) isolates that appears to serve as a platform for resistance genes and may have virulence-related functions as well. Carriage of antibiotic resistance genes on transposable elements and mobile plasmids in commensal E. coli renders the resistome highly dynamic. IMPORTANCE Rising antibiotic resistance in human-associated bacterial pathogens is a serious threat to our ability to treat many infectious diseases. It is critical to understand how acquired resistance genes move in and through bacteria associated with humans, particularly for species such as Escherichia coli that are very common in the human gut but can also be dangerous pathogens. This work combined two distinct DNA sequencing approaches to allow us to explore the genomes of E. coli from college students to show that the antibiotic resistance genes these bacteria have acquired are usually carried on a specific type of plasmid that is naturally transferrable to other E. coli, and likely to other related bacteria.

scholarly journals Enzymatic Synthesis and Functional Characterization of Bioactive Microcin C-Like Compounds with Altered Peptide Sequence and Length

2015 ◽  
Vol 197 (19) ◽  
pp. 3133-3141 ◽  
Author(s):  
Olga Bantysh ◽  
Marina Serebryakova ◽  
Inna Zukher ◽  
Alexey Kulikovsky ◽  
Darya Tsibulskaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Functional Characterization ◽  
Trna Synthetase ◽  
Biologically Active ◽  
Peptide Sequence ◽  
Target Cells ◽  
Content Type ◽  
Peptide Length

ABSTRACTEscherichia colimicrocin C (McC) consists of a ribosomally synthesized heptapeptide attached to a modified adenosine. McC is actively taken up by sensitiveEscherichia colistrains through the YejABEF transporter. Inside the cell, McC is processed by aminopeptidases, which release nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC is synthesized by the MccB enzyme, which terminally adenylates the MccA heptapeptide precursor MRTGNAN. Earlier, McC analogs with shortened peptide lengths were prepared by total chemical synthesis and were shown to have strongly reduced biological activity due to decreased uptake. Variants with longer peptides were difficult to synthesize, however. Here, we used recombinant MccB to prepare and characterize McC-like molecules with altered peptide moieties, including extended peptide lengths. We find that N-terminal extensions ofE. coliMccA heptapeptide do not affect MccB-catalyzed adenylation and that some extended-peptide-length McC analogs show improved biological activity. When the peptide length reaches 20 amino acids, both YejABEF and SbmA can perform facilitated transport of toxic peptide adenylates inside the cell. A C-terminal fusion of the carrier maltose-binding protein (MBP) with the MccA peptide is also recognized by MccBin vivoandin vitro, allowing highly specific adenylation and/or radioactive labeling of cellular proteins.IMPORTANCEEnzymatic adenylation of chemically synthesized peptides allowed us to generate biologically active derivatives of the peptide-nucleotide antibiotic microcin C with improved bioactivity and altered entry routes into target cells, opening the way for development of various McC-based antibacterial compounds not found in nature.

scholarly journals Escherichia coli and Klebsiella pneumoniae Producing CTX-M Cephalosporinase from Swine Finishing Barns and Their Association with Antimicrobial Use

2012 ◽  
Vol 79 (3) ◽  
pp. 1052-1054 ◽  
Author(s):  
Dixie F. Mollenkopf ◽  
Jennifer M. Mirecki ◽  
Joshua B. Daniels ◽  
Julie A. Funk ◽  
Steven C. Henry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Antimicrobial Use ◽  
Fecal Samples ◽  
Content Type ◽  
Extended Spectrum ◽  
Lactamase Gene

ABSTRACTWe report the recovery ofEscherichia coliorKlebsiella pneumoniaecontaining the extended-spectrum β-lactamase geneblaCTX-Mfrom 24 of 1,495 (1.6%) swine fecal samples in 8 of 50 (16%) finishing barns located in 5 U.S. states. We did not detect an association between antimicrobial use and recovery ofblaCTX-M.

scholarly journals Characterization of CIA-1, an Ambler Class A Extended-Spectrum β-Lactamase from Chryseobacterium indologenes

2011 ◽  
Vol 56 (1) ◽  
pp. 588-590 ◽  
Author(s):  
Takehisa Matsumoto ◽  
Mika Nagata ◽  
Nau Ishimine ◽  
Kenji Kawasaki ◽  
Kazuyoshi Yamauchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reference Strain ◽  
Content Type ◽  
Chryseobacterium Indologenes ◽  
Esbl Gene ◽  
Class A ◽  
Extended Spectrum ◽  
Class B ◽  
Lactamase Gene

ABSTRACTAn Ambler class A β-lactamase gene,blaCIA-1, was cloned from the reference strainChryseobacterium indologenesATCC 29897 and expressed inEscherichia coliBL21. TheblaCIA-1gene encodes a novel extended-spectrum β-lactamase (ESBL) that shared 68% and 60% identities with the CGA-1 and CME-1 β-lactamases, respectively.blaCIA-1-like genes were detected from clinical isolates. In addition to the metallo-β-lactamase IND of Ambler class B,C. indologeneshas a class A ESBL gene,blaCIA-1, located on the chromosome.

scholarly journals Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Eduardo Soto ◽  
Norma Espinosa ◽  
Miguel Díaz-Guerrero ◽  
Meztlli O. Gaytán ◽  
José L. Puente ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Diarrheal Disease ◽  
Functional Characterization ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Content Type ◽  
Enterocyte Effacement

ABSTRACT The type III secretion system (T3SS) is a supramolecular machine used by many bacterial pathogens to translocate effector proteins directly into the eukaryotic host cell cytoplasm. Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrheal disease in underdeveloped countries. EPEC virulence relies on a T3SS encoded within a chromosomal pathogenicity island known as the locus of enterocyte effacement (LEE). In this work, we pursued the functional characterization of the LEE-encoded protein EscK (previously known as Orf4). We provide evidence indicating that EscK is crucial for efficient T3S and belongs to the SctK (OrgA/YscK/MxiK) protein family, whose members have been implicated in the formation of a sorting platform for secretion of T3S substrates. Bacterial fractionation studies showed that EscK localizes to the inner membrane independently of the presence of any other T3SS component. Combining yeast two-hybrid screening and pulldown assays, we identified an interaction between EscK and the C-ring/sorting platform component EscQ. Site-directed mutagenesis of conserved residues revealed amino acids that are critical for EscK function and for its interaction with EscQ. In addition, we found that T3S substrate overproduction is capable of compensating for the absence of EscK. Overall, our data suggest that EscK is a structural component of the EPEC T3SS sorting platform, playing a central role in the recruitment of T3S substrates for boosting the efficiency of the protein translocation process. IMPORTANCE The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

scholarly journals Complete Nucleotide Sequence of an IncI2 Plasmid CoharboringblaCTX-M-55andmcr-1

2016 ◽  
Vol 60 (8) ◽  
pp. 5014-5017 ◽  
Author(s):  
Jian Sun ◽  
Xing-Ping Li ◽  
Run-Shi Yang ◽  
Liang-Xing Fang ◽  
Wei Huo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Complete Nucleotide Sequence ◽  
Sequence Similarity ◽  
Conjugative Plasmid ◽  
Insertion Sequences ◽  
Content Type ◽  
Extensive Sequence ◽  
Extended Spectrum ◽  
Lactamase Gene

ABSTRACTWe report the complete nucleotide sequence of a plasmid, pA31-12, carryingblaCTX-M-55andmcr-1from a chickenEscherichia coliisolate. pA31-12 has an IncI2 replicon that displays extensive sequence similarity with pHN1122-1-borneblaCTX-M-55and pHNSHP45-bornemcr-1. Insertion sequences ISEcp1and ISApl1are responsible for the mobilization ofblaCTX-M-55andmcr-1, respectively. The colocalization ofmcr-1with an extended-spectrum β-lactamase gene on a conjugative plasmid may accelerate the dissemination of both genes by coselection.

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