scholarly journals Characterization of Extended-Spectrum Cephalosporin (ESC) Resistance in Salmonella Isolated from Chicken and Identification of High Frequency Transfer of blaCMY-2 Gene Harboring Plasmid In Vitro and In Vivo

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1778
Author(s):  
Bo-Ram Kwon ◽  
Bai Wei ◽  
Se-Yeoun Cha ◽  
Ke Shang ◽  
Jun-Feng Zhang ◽  
...  
Keyword(s):  
High Frequency ◽  
Direct Evidence ◽  
E Coli ◽  
Chicken Isolate ◽  
Chicken Feces ◽  
Mouse Intestine ◽  
Meat Samples

A total of 136 Salmonella isolates from chicken feces and meat samples of the top 12 integrated chicken production companies throughout Korea were collected. Among the 17 ESC-resistant Salmonella; blaCTX-M-15 was the most prevalent gene and two strains carried blaTEM-1/blaCTX-M-15 and blaCMY-2, respectively. The transferable blaCTX-M-15 gene was carried by IncFII plasmid in three isolates and the blaCMY-2 gene carried by IncI1 plasmid in one isolate. blaCMY-2 gene-harboring strain was selected as the donor based on the high frequency of blaCMY-2 gene transfer in vitro and its transfer frequencies were determined at 10−3 transconjugants per recipient. The transfer of blaCMY-2 gene-harboring plasmid derived from chicken isolate into a human pathogen; enteroinvasive Escherichia coli (EIEC), presented in mouse intestine with about 10−1 transfer frequency without selective pressure. From the competition experiment; blaCMY-2 gene-harboring transconjugant showed variable fitness burden depends on the parent strains. Our study demonstrated direct evidence that the blaCMY-2 gene harboring Salmonella from chicken could frequently transfer its ESC-resistant gene to E. coli in a mouse intestine without antimicrobial pressure; resulting in the emergence of multidrug resistance in potentially virulent EIEC isolates of significance to human health; which can increase the risk of therapeutic inadequacy or failures.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

Characterization of in vitro and in vivo murine models of human enteropathogenic E. coli (EPEC) infection

2003 ◽  
Vol 124 (4) ◽  
pp. A558
Author(s):  
Suzana D. Savkovic ◽  
Farol L. Tomson ◽  
Michelle Muza ◽  
Gail Hecht
Keyword(s):  
Murine Models ◽  
E Coli

scholarly journals Characterization of MazFSa, an Endoribonuclease from Staphylococcus aureus

2007 ◽  
Vol 189 (24) ◽  
pp. 8871-8879 ◽  
Author(s):  
Zhibiao Fu ◽  
Niles P. Donegan ◽  
Guido Memmi ◽  
Ambrose L. Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Environmental Stress Response ◽  
Binding Studies ◽  
Cell Growth Arrest ◽  
Consensus Sequences ◽  
E Coli ◽  
Endoribonuclease Activity

ABSTRACT The mazEF homologs of Staphylococcus aureus, designated mazEFsa , have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF Sa , as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF Sa leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF Sa is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF Sa cleaves single-strand RNA preferentially at the 5′ side of the first U or 3′ side of the second U residue within the consensus sequences VUUV′ (where V and V′ are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE Sa binds MazF Sa to form a complex to inhibit the endoribonuclease activity of MazF Sa . Contrary to the system in E. coli, exposure to selected antibiotics augmented mazEFsa transcription, akin to what one would anticipate from the environmental stress response of the sigB system. These data indicate that the mazEF system of S. aureus differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.

Characterization of P. sativum chloroplast psbA transcripts produced in vivo, in vitro and in E. coli

1986 ◽  
Vol 6 (4) ◽  
pp. 229-243 ◽  
Author(s):  
Scott K. Boyer ◽  
John E. Mullet
Keyword(s):  
E Coli

scholarly journals Identification and Partial Characterization of an L-Tyrosine Aminotransferase (TAT) fromArabidopsis thaliana

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Pranav R. Prabhu ◽  
André O. Hudson
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Family ◽  
Tyrosine Aminotransferase ◽  
Functional Complementation ◽  
E Coli ◽  
Full Length Cdna ◽  
Putative Aminotransferase

The aminotransferase gene family in the model plantArabidopsis thalianaconsists of 44 genes. Twenty six of these enzymes are classified as characterized meaning that the reaction(s) that the enzyme catalyzes are documented using experimental means. The remaining 18 enzymes are uncharacterized and are therefore deemed putative. Our laboratory is interested in elucidating the function(s) of the remaining putative aminotransferase enzymes. To this end, we have identified and partially characterized an aminotransferase (TAT) enzyme from Arabidopsis annotated by the locus tag At5g36160. The full-length cDNA was cloned and the purified recombinant enzyme was characterized usingin vitroandin vivoexperiments.In vitroanalysis showed that the enzyme is capable of interconverting L-Tyrosine and 4-hydroxyphenylpyruvate, and L-Phenylalanine and phenylpyruvate.In vivoanalysis by functional complementation showed that the gene was able to complement anE. coliwith a background of aminotransferase mutations that confers auxotrophy for L-Tyrosine and L-Phenylalanine.

scholarly journals Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies

Cancers ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 414 ◽  
Author(s):  
Radhia M’kacher ◽  
Monika Frenzel ◽  
Mustafa Al Jawhari ◽  
Steffen Junker ◽  
Corina Cuceu ◽  
...  
Keyword(s):  
Animal Model ◽  
Histone Deacetylase ◽  
High Frequency ◽  
Histone Deacetylase Inhibitor ◽  
Telomerase Activity ◽  
Nsg Mice ◽  
Deacetylase Inhibitor

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30−/CD15− cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (−/−)(NSG) mice. Using cell sorting, we demonstrate that CD30−/CD15− subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30−/CD15− cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 103 cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30−/CD15− cells exhibiting high telomerase activity and telomere dysfunction.

scholarly journals Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

2005 ◽  
Vol 389 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Rajesh K. Soni ◽  
Parul Mehra ◽  
Gauranga Mukhopadhyay ◽  
Suman Kumar Dhar
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dnab Helicase ◽  
H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

scholarly journals Truncation of poly-N-acetylglucosamine (PNAG) polymerization with N-acetylglucosamine analogues

2021 ◽  
Author(s):  
Zachary Morrison ◽  
Alexander Eddenden ◽  
Adithya S Subramanian ◽  
P. Lynne Howell ◽  
mark nitz
Keyword(s):  
Biofilm Formation ◽  
Chain Termination ◽  
Salvage Pathway ◽  
Biofilm Inhibition ◽  
E Coli ◽  
Substrate Analogues

Bacteria require polysaccharides for structure, survival, and virulence. Despite the central role these structures play in microbiology few tools are available to manipulate their production. In E. coli the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro the inhibitors cause PNAG chain termination; consistent with the mechanism of PNAG polymerization from the non-reducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

scholarly journals Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

2020 ◽  
Author(s):  
Fabienne Benz ◽  
Jana S. Huisman ◽  
Erik Bakkeren ◽  
Joana A. Herter ◽  
Tanja Stadler ◽  
...  
Keyword(s):  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Transfer Genes ◽  
Serovar Typhimurium ◽  
Mouse Intestine ◽  
Specific Factors ◽  
Natural Strains

Abstract Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.

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