scholarly journals 1127. Genomic Analysis of Biofilm-Forming Enteroinvasive E. coli Emergent Pathogen

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S337-S338
Author(s):  
Oscar Gomez-Duarte ◽  
Julio Guerra ◽  
Ricky Ko
Keyword(s):  
Virulence Factors ◽  
Dna Sequences ◽  
Horizontal Transfer ◽  
Antibiotic Resistance Genes ◽  
Genomic Analysis ◽  
Coli Strain ◽  
Virulence Plasmid ◽  
E Coli ◽  
Query Coverage

Abstract Background Enteroinvasive Escherichia coli (EIEC) are involved in dysenteric diarrhea among children in low- and middle-income countries. EIEC strains isolated in Colombia, South America were shown to form biofilms and to be invasive in vitro. The O96:H19 serotypes and biofilm formation (BF) are not common phenotypes among EIEC, and the role they may play in diarrhea is at present unknown. The main goal of this study was to identify virulence and BF genes from EIEC genomic data. We hypothesize that EIEC O96:H19 strain 52.1 originated from horizontal transfer of a Shigella-like virulence plasmid into a non-EIEC pathogenic E coli strain. Methods WGS was performed on the BF-EIEC 52.1 strain using NextGen Illumina and Pacific Biosciences (PacBio) platforms. Publically available genomes from other EIEC O96H19 and Shigella genomes previously published were analyzed using online available software and databases including NCBI, BLAST, Mauve, among others. This analysis was tailored to identify virulence factors from the virulence factor database (VFDB). BLASTn was used to determine identity and query coverage of genes encoding the Shigella virulence factors. EIEC and Shigella genomes were analyzed on a multiple genome alignment software (Mauve) to verify results from BLASTn and to determine pseudogenes. Results The genome of EIEC O96:H19 strain 52.1 was 5,193,449 bp in size, containing 5,050 coding DNA sequences (CDSs). O96:H19 strain 52.1 carries three plasmids, the invasion plasmid (pINV) contains all type 3 secretion system (TTSS) and TTSS effectors genes previously described for Shigella and EIEC O96:H19 CFSAN029787 Italian strain. Non-TTSS virulence genes were also identified, including: long polar fimbrial gene (IpfA), enterotoxin (senB), and antibiotic resistance genes. Conclusion The EIEC O96:H19 strain 52.1 genome carries TTSS genes within a virulence plasmid, protein effector genes, and enterotoxin genes known to be associated with EIEC virulence. The EIEC O96:H19 stain 52.1 is an emergent diarrheagenic pathogen likely derived from an E. coli O96:H19 strain that acquired a Shigella-like virulence plasmid by horizontal transfer. Disclosures All authors: No reported disclosures.

scholarly journals Obtaining of the recombinant Rhil7-Cbd fusion protein

2020 ◽  
Vol 26 ◽  
pp. 282-286
Author(s):  
M. O. Usenko ◽  
O. B. Gorbatiuk ◽  
O. V. Okunev ◽  
D. M. Irodov ◽  
M. V. Koval’chuk ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Fusion Protein ◽  
Dna Sequences ◽  
Inclusion Bodies ◽  
Plasmid Vector ◽  
Coli Strain ◽  
Iptg Induction ◽  
E Coli ◽  
Interleukin 7

Aim. The purpose was to obtain the recombinant fusion protein based on the human interleukin-7 (rhIL7) and cellulose binding domain (CBD). Methods. The DNA sequences encoding rhIL-7 and CBD were subcloned into the pET24a(+). Vector containing the 6His-tag sequence for further chromatographic purification of the target protein. The cells of E. coli strain BL21(DE3) were transformed with pET24-rhIL7-CBD plasmid vector. Protein synthesis was induced in clones by IPTG and by autoinduction. Results. An expression cassette of rhIL7-CBD protein has been designed. Producers of rhIL7-CBD protein were obtained. It was shown that the autoinduction protocol provides protein synthesis in E. coli (but IPTG induction doesn’t). Protein was obtained in the cytoplasmic fraction in form of inclusion bodies. In vitro method of purification of rhIL7-CBD from inclusion bodies has been worked out. Conclusions. The obtained rhIL7-CBD protein can be used for the microcrystalline cellulose immunosorbent construction for the purification of the specific polyclonal IL-7 antibodies and also for qualitative and quantitative analysis of IL-7 receptors. Keywords: IL-7, CBD, inclusion bodies, renaturation.

scholarly journals In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaitlin S. Witherell ◽  
Jason Price ◽  
Ashok D. Bandaranayake ◽  
James Olson ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
E Coli ◽  
Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

Studies of Virulence Factors of Avian Pathogenic Escherichia coli in Avian Colibacillosis by In vitro Method and PCR

2021 ◽  
Author(s):  
Ayushi Singh ◽  
Daljeet Chhabra ◽  
Rakhi Gangil ◽  
Rakesh Sharda ◽  
Ravi Sikrodia ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Molecular Detection ◽  
Bacterial Disease ◽  
Avian Pathogenic Escherichia Coli ◽  
E Coli ◽  
Pathological Conditions ◽  
Biofilm Production ◽  
Pathogenic Escherichia Coli ◽  
Stx1 Gene

Background: Avian colibacillosis is considered as major cause of morbidity and mortality in poultry. It is a common bacterial disease of poultry and many virulence factors of E. coli are associated with the disease. The current study was aimed to investigate the presence of some virulence factors of E. coli isolated from the cases of colibacillosis.Methods: In present study, total 150 samples (liver, heart, lungs, air sacs and feaces) of chicken exhibiting pathological conditions of colibacillosis were collected from various poultry farms (organized and backyard) situated in and around Mhow and Indore cities. E.coli was isolated and identified from the samples on the basis of cultural characteristics and biochemical test. All E. coli isolates were further subjected to evaluate the presence of virulence factors such as biofilm production, haemolysis, invasiveness and molecular detection of fimH and stx1 gene.Result: Out of these 51.33% of incidence of E. coli was recorded. E. coli O84 and O149 serotypes were found most prevalent. Out of 77 isolates, 46 (59.7%) and 45 (58.4%) were positive for biofilm formation by tube method and modified CRA method, respectively. All E. coli isolates were showing invasiveness in congo red binding assay while none of the isolates was found haemolytic. Molecular detection revealed the presence of fimH (508bp) gene in 33.3% of tested samples while stx1 gene could not be detected in any isolates.

scholarly journals The Production of the Recombinant Protein of Anthrax Bacteriolysine PlyPH and In Vitro Study of the Lytic Properties of the Protein Coded for them Against Bacillus anhracis Microbial Cells

2019 ◽  
pp. 5-22
Author(s):  
Onuchina N.V., Soybanov V.D.
Keyword(s):  
In Vitro Study ◽  
Coli Strain ◽  
Vitro Study ◽  
Chromosomal Dna ◽  
Lytic Enzymes ◽  
Microbial Cells ◽  
Phage Gene ◽  
E Coli ◽  
Species Specific

The causative agent of anthrax - Bacillus anthracis, due to the prevalence of its natural foci in Russia, high virulence for humans and most mammals, the unique resistance of spore forms to environmental factors and repeated use in terrorist acts, is an extremely dangerous biological agent. Therefore, the search for new effective drugs for the diagnosis and treatment of anthrax, including diseases caused by antibiotic-resistant strains of B. anthracis is necessary. The use of lytic enzymes of species-specific bacteriophages is a new trend in the diagnosis, prevention and treatment of infectious diseases. The goal of this work is the cloning of the anthrax bacteriolysin PlyPH gene as part of the pTrcHis2C vector in Escherichia coli and the in vitro study of the lytic properties of the protein encoded by it against B. anthracis microbial cells. According to the complete sequencing of the B. anthracis genomes of the Ames, Stern 34F2 and JB17 strains, a prophage was found in their chromosomal DNA, which lost part of the structural genes necessary for its replication, but retained a gene with a high degree of homology with the bacteriolysin γ phage gene. For amplification and subsequent cloning of the PlyPH gene, we developed primers containing EcoRI and BamHI restriction enzyme recognition sites. Amplification of the PlyPH gene in a polymerase chain reaction (PCR) with a developed pair of primers was performed using the Stern 34F2 strain of the anthrax microbe as a template. Based on the obtained amplification products and the pTrcHis2C vector, we constructed a recombinant plasmid containing the bacteriolysin synthesis PlyPH gene and stably functioning in the cells of the recombinant E. coli strain. In the course of research, it has been established that microbial cells of the E. coli recombinant TOP10 strain provide for the production of the bacteriolysin of the anthrax prophage, PlyPH , which has the ability to in vitro lyse the vegetative cells of the STI-1 vaccine strain of B. anthracis

scholarly journals Synthesis of silver nanoparticles from extracts of Scytonema geitleri HKAR-12 and their in vitro antibacterial and antitumor potentials

2019 ◽  
Vol 8 (3) ◽  
pp. 576-585
Keyword(s):  
Silver Nanoparticles ◽  
Coli Strain ◽  
X Ray Diffraction ◽  
Xrd Pattern ◽  
E Coli ◽  
Vis Spectroscopy ◽  
Shape Structure ◽  
Actual Size

In the present study silver nanoparticles (AgNPs) have been synthesized through the cell-free extracts of the rooftop dwelling cyanobacterium Scytonema geitleri HKAR-12. UV-VIS spectroscopy, FTIR, X-ray diffraction, SEM and TEM were used for the determination of morphological, structural and optical properties of synthesized AgNPs. Extracts of Scytonema geitleri HKAR-12 have the ability to reduce AgNO3 to Ag0. Sharp peak at 422 nm indicated the rapid synthesis of AgNPs. FTIR results showed the presence of different groups responsible for the reduction of AgNO3 to AgNPs. XRD pattern confirmed the crystalline nature of AgNPs. SEM showed the bead shape structure of AgNPs. TEM confirmed the actual size of AgNPs to be ranging between 9-17 nm. AgNPs showed antibacterial activity against Pseudomonas aeruginosa, Escherichia coli strain1 and E. coli strain 2 and 11 μg/mL of AgNPs effectively inhibited the growth of MCF-7 cells. Hence, Scytonema geitleri HKAR-12, isolated from the rooftop could serve as a desirable biological candidate for convenient and cheap production of AgNPs having antimicrobial and anti-cancerous properties.

scholarly journals 702. Zinc Blockade of SOS Response Inhibits Horizontal Transfer of Antibiotic Resistance Genes in Enteric Bacteria

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S253-S253
Author(s):  
John Crane ◽  
Mark Sutton ◽  
Muhammad Cheema ◽  
Michael Olyer
Keyword(s):  
Dna Damage ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Horizontal Transfer ◽  
Antibiotic Resistance Genes ◽  
Sos Response ◽  
Enteric Bacteria ◽  
Extracellular Dna ◽  
In Vitro Assays ◽  
E Coli

Abstract Background The SOS response is a conserved response to DNA damage that is found in Gram negative and Gram-positive bacteria. When DNA damage is sustained and severe, activation of error-prone DNA polymerases can induce a higher mutation rate then normally observed, which is called the mutator phenotype or hypermutation. We previously showed that zinc blocked the hypermutation response induced by quinolone antibiotics and mitomycin C in E. coli and Klebsiella pneumoniae (Bunnell BE, Escobar JF, Bair KL, Sutton MD, Crane JK (2017). Zinc blocks SOS-induced antibiotic resistance via inhibition of RecA in Escherichia coli. PLoS ONE 12(5): e0178303. https://doi.org/10.1371/journal.pone.0178303.) In addition to causing copying errors in DNA replication, Beaber et al. showed that induction of the SOS response increased the frequency of horizontal gene transfer into Vibrio cholerae, an organism naturally competent at uptake of extracellular DNA. (Beaber JW, Hochhut B, Waldor MK. 2003. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427:72–74.) Methods. In this study, we tested whether induction of the SOS response could induce transfer of antibiotic resistance from Enterobacter cloacae into E. coli, and whether zinc could inhibit that inter-species transfer of antibiotic resistance. Results. Ciprofloxacin, an inducer of the SOS response, increased the rate of transfer of an extended spectrum β-lactamase (ESBL) gene from Enterobacter into a susceptible E. coli strain. Zinc blocked SOS-induced horizontal transfer of §-lactamase into E. coli. Other divalent metals, such as iron and manganese, failed to inhibit these responses. Conclusion. In vitro assays showed that zinc blocked the ability of RecA to bind to ssDNA, an early step in the SOS response, suggesting the mechanism by which zinc blocks the SOS response. Disclosures All authors: No reported disclosures.

scholarly journals Transferable Extended-Spectrum β-Lactamase (ESBL) Plasmids in Enterobacteriaceae from Irrigation Water

2020 ◽  
Vol 8 (7) ◽  
pp. 978
Author(s):  
Maria-Theresia Gekenidis ◽  
Anita Kläui ◽  
Kornelia Smalla ◽  
David Drissner
Keyword(s):  
Irrigation Water ◽  
Antibiotic Resistance Genes ◽  
Resistant Bacteria ◽  
Edible Plant ◽  
Transfer Resistance ◽  
Phenotypic Resistance ◽  
E Coli ◽  
Plant Parts ◽  
Extended Spectrum

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are classified as serious threats to human health by the U.S. Centers for Disease Control and Prevention. Water used for irrigation of fresh produce can transmit such resistant bacteria directly to edible plant parts. We screened ESBL-producing Escherichia coli, Enterobacter cloacae, and Citrobacter freundii isolated from irrigation water for their potential to transmit resistance to antibiotic-susceptible E. coli. All strains were genome-sequenced and tested in vitro for transmission of resistance to third-generation cephalosporins on solid agar as well as in liquid culture. Of the 19 screened isolates, five ESBL-producing E. coli were able to transfer resistance with different efficiency to susceptible recipient E. coli. Transconjugant strains were sequenced for detection of transferred antibiotic resistance genes (ARGs) and compared to the known ARG pattern of their respective donors. Additionally, phenotypic resistance patterns were obtained for both transconjugant and corresponding donor strains, confirming ESBL-producing phenotypes of all obtained transconjugants.

scholarly journals The Bacterial DNA Binding Protein MatP Involved in Linking the Nucleoid Terminal Domain to the Divisome at Midcell Interacts with Lipid Membranes

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Begoña Monterroso ◽  
Silvia Zorrilla ◽  
Marta Sobrinos-Sanguino ◽  
Miguel Ángel Robles-Ramos ◽  
Carlos Alfonso ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Lipid Membranes ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Content Type ◽  
E Coli ◽  
Daughter Cells ◽  
Z Ring

ABSTRACTDivision ring formation at midcell is controlled by various mechanisms inEscherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipidsin vitro. We found that, when encapsulated inside vesicles and microdroplets generated by microfluidics, MatP accumulates at phospholipid bilayers and monolayers matching the lipid composition in theE. coliinner membrane. MatP binding to lipids was independently confirmed using lipid-coated microbeads and biolayer interferometry assays, which suggested that the recognition is mainly hydrophobic. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein, but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the association of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.IMPORTANCEThe division of anE. colicell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integratedin vivoandin vitroanalysis provides evidence that MatP can interact with lipid membranes reproducing the phospholipid mixture in theE. coliinner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

Extended-spectrum resistance to β-lactams/β-lactamase inhibitors (ESRI) evolved from low-level resistant Escherichia coli

2019 ◽  
Author(s):  
Ángel Rodríguez-Villodres ◽  
María Luisa Gil-Marqués ◽  
Rocío Álvarez-Marín ◽  
Rémy A Bonnin ◽  
María Eugenia Pachón-Ibáñez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clavulanic Acid ◽  
Genomic Analysis ◽  
E Coli ◽  
Extended Spectrum ◽  
Copy Numbers ◽  
Resistance Patterns ◽  
Amoxicillin Clavulanic Acid

Abstract Objectives Escherichia coli is characterized by three resistance patterns to β-lactams/β-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood. Methods Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of β-lactamase was quantified in these isolates. Results We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing β-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure. Conclusions This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs.

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