We describe a brand new set of instruments for inserting DNA into the bacterial chromosome. The system makes use of site-specific recombination reactions carried out by bacteriophage integrases to combine plasmids at as much as eight phage attachment websites in E. coli MG1655. The introduction of mutant loxP websites within the integrating plasmids permits repeated elimination of antibiotic resistance genes and different plasmid sequences with out hazard of inducing chromosomal rearrangements. The protocol for Cre-mediated antibiotic resistance gene elimination is drastically simplified by introducing the Cre plasmid by phage an infection. Lastly, we’ve additionally developed a set of 4 independently inducible expression modules with tight management and excessive dynamic vary which may be inserted at particular chromosomal places.
Qac efflux pumps from proteobacterial multidrug-resistant plasmids are integron-encoded and confer resistance to quaternary ammonium compound (QAC) antiseptics, nevertheless, many are uncharacterized and misannotated. A survey of >2000 plasmid-encoded qac recognized 37 distinctive qac sequences that correspond to one among 5 consultant motifs: QacE, QacEΔ1, QacF/L, QacH/I, and QacG. Antimicrobial susceptibility testing of every cloned qac member in Escherichia coli, highlighted distinctive antiseptic susceptibility patterns that have been most outstanding when cells grew as biofilms.
Fosfomycin is a vital antibiotic for the therapy of MDR Enterobacteriaceae infections. Excessive susceptibility charges are, nevertheless, threatened by the unfold of plasmids encoding fosfomycin-modifying enzymes. On this research, we sought to characterize the genetic context of fosA in plasmids from Escherichia coli and Klebsiella spp. isolates recovered from meals, wastewater and floor water in Switzerland. vE. coli and Klebsiella spp. isolates collected between 2012 and 2019 in Switzerland have been screened for fosfomycin resistance. Presence of fosA was verified by PCR and sodium phosphonoformate (PPF) disc potentiation testing, and transferability was examined utilizing conjugation assays. Entire-genome sequences together with full fosA-containing plasmids have been decided utilizing long- and short-read sequencing.
Switch of Antibiotic Resistance Plasmid from Commensal E. coli In the direction of Human Intestinal Microbiota within the M-SHIME: Effect of E. coli dosis, Human Particular person and Antibiotic Use
Together with (in) direct contact with animals and a contaminated setting, people are uncovered to antibiotic-resistant micro organism by consumption of meals. The implications of ingesting antibiotic-resistant commensal micro organism are unknown, as dose-response information on resistance switch and spreading in our intestine is missing. On this research, switch of a resistance plasmid (IncF), harbouring a number of antibiotic resistance genes, from a commensal E. coli pressure in the direction of human intestinal microbiota was assessed utilizing a Mucosal Simulator of the Human Intestinal Ecosystem (M-SHIME).
Extra particularly, the impact of the preliminary E. coli plasmid donor focus (105 and 107 CFU/meal), antibiotic therapy (cefotaxime) and human particular person (n = 6) on plasmid switch in the direction of lumen coliforms and anaerobes was decided. Switch of the resistance plasmid to luminal coliforms and anaerobes was noticed shortly after the donor pressure arrived within the colon and was unbiased of the ingested dose. Switch occurred in all six simulated colons and regardless of their distinctive microbial group composition, no variations might be detected in antibiotic resistance switch charges between the simulated human colons. Presence of the resistance plasmid was confirmed and quantified by PCR and qPCR. Cefotaxime therapy led to a big discount (85%) in resistant coliforms, nevertheless no important impact on the entire variety of cultivable coliforms and anaerobes was noticed.
Translesion synthesis (TLS) is an occasion to deal with DNA damages. Throughout TLS, the accountable TLS polymerase often elicits untargeted mutagenesis as doubtlessly a supply of genetic range. Figuring out such untargeted mutations in vivo is difficult because of the bulk of DNA that doesn’t endure TLS. Right here, we current a protocol to complement a plasmid pool that underwent Pol V-mediated TLS in Escherichia coli for mass sequencing. The idea of this protocol might be utilized into any species. For full particulars on the use and execution of this protocol, please confer with Isogawa et al. (2018).
Plasmid Vectors for in Vivo Choice-Free Use with the Probiotic E. coli Nissle 1917
Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium, generally employed to deal with sure gastrointestinal problems. It’s quick rising as an vital goal for the event of therapeutic engineered micro organism, benefiting from the wealth of data of E. coli biology and ease of manipulation. Bacterial artificial biology tasks generally make the most of engineered plasmid vectors, that are easy to engineer and may reliably obtain excessive ranges of protein expression. Nonetheless, plasmids usually require antibiotics for upkeep, and the administration of an antibiotic is usually incompatible with in vivo experimentation or therapy. EcN natively accommodates plasmids pMUT1 and pMUT2, which haven’t any identified operate however are secure throughout the micro organism.
Right here, we describe the event of the pMUT plasmids into a strong platform for engineering EcN for in vivo experimentation, alongside a CRISPR-Cas9 system to take away the native plasmids. We systematically engineered each pMUT plasmids to include choice markers, fluorescent markers, temperature delicate expression, and curli secretion programs to export a customizable purposeful materials into the extracellular area.
We then exhibit that the engineered plasmids have been maintained in micro organism because the engineered micro organism cross by way of the mouse GI tract with out choice, and that the secretion system stays purposeful, exporting functionalized curli proteins into the intestine. Our plasmid system presents a platform for the fast improvement of therapeutic EcN micro organism.