Experimental Simulation of the Effects of an Initial Antibiotic Treatment on a Subsequent Treatment after Initial Therapy Failure

General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Received: 24 October 2013; in revised form: 23 January 2014 / Accepted: 29 January 2014 / Published: 17 February 2014 Abstract: Therapy failure of empirical antibiotic treatments prescribed by primary care physicians occurs commonly. The effect of such a treatment on the susceptibility to second line antimicrobial drugs is unknown. Resistance to amoxicillin was rapidly induced or selected in E. coli at concentrations expected in the patient's body. Strains with reduced susceptibility outcompeted the wild-type whenever antibiotics were present, even in low concentrations that did not affect the growth rates of both strains. Exposure of E. coli to amoxicillin caused moderate resistance to cefotaxime. The combined evidence suggests that initial treatment by amoxicillin has a negative effect on subsequent therapy with beta-lactam antibiotics

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes

Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells

Compensation of the Metabolic Costs of Antibiotic Resistance by Physiological Adaptation in Escherichia coli

Antibiotic resistance is often associated with metabolic costs. To investigate the metabolic consequences of antibiotic resistance, the genomic and transcriptomic profiles of an amoxicillin-resistant Escherichia coli strain and the wild type it was derived from were compared. A total of 125 amino acid substitutions and 7 mutations that were located <1,000 bp upstream of differentially expressed genes were found in resistant cells. However, broad induction and suppression of genes were observed when comparing the expression profiles of resistant and wild-type cells. Expression of genes involved in cell wall maintenance, DNA metabolic processes, cellular stress response, and respiration was most affected in resistant cells regardless of the absence or presence of amoxicillin. The SOS response was downregulated in resistant cells. The physiological effect of the acquisition of amoxicillin resistance in cells grown in chemostat cultures consisted of an initial increase in glucose consumption that was followed by an adaptation process. Furthermore, no difference in maintenance energy was observed between resistant and sensitive cells. In accordance with the transcriptomic profile, exposure of resistant cells to amoxicillin resulted in reduced salt and pH tolerance. Taken together, the results demonstrate that the acquisition of antibiotic resistance in E. coli is accompanied by specifically reorganized metabolic networks in order to circumvent metabolic costs. The overall effect of the acquisition of resistance consists not so much of an extra energy requirement, but more a reduced ecological range

Minimum inhibitory concentration (MIC), maximum growth rate (μ_max) and β-lactamase activity of acceptor, donor and transconjugant cells.

<p>^a Average maximum growth rate of three transconjugants randomly chosen from three different transfer experiments. All individual transconjugants were grown in two replicates.</p><p>^b Specific activity is reported in nanomoles of nitrocefin hydrolyzed per minute per milligram of protein. The results are presented as the means and standard deviations of two independent measurements.</p><p>^c β-lactamase activity was averaged from transconjugants obtained in two individual experiments</p><p>Minimum inhibitory concentration (MIC), maximum growth rate (μ_max) and β-lactamase activity of acceptor, donor and transconjugant cells.</p

Transfer of pESBL-283 from ESBL242 (donor, amp^R) to <i>E</i>. <i>coli</i> MG1655 (acceptor, chlor^R).

<p>After donor and acceptor cells reached steady states in separate chemostats, cultures were mixed in a ratio of 1:1 in an empty reactor vessel and immediately supplied with fresh medium at the same dilution rate. Experimental conditions in each of the panels: (A) 0 μg/ml at D = 0.2 h^-1, (B) 512 μg/ml ampicillin at D = 0.2 h^-1, (C) 0 μg/ml at D = 0.4 h^-1 and (D) 512 μg/ml ampicillin at D = 0.4 h^-1.</p

Number of transconjugants during continuous cultivation of ESBL242 (donor) and E. coli MG1655 (acceptor) cells at t = 6h or 24h.

<p><i>E</i>. <i>coli</i> MG1655 and ESBL242 cells were cultivated separately and continuously at a dilution rate of D = 0.2 or 0.4 h^-1 and subsequently mixed with a ratio of 1:1 (t = 0h).</p

Plasmids found in the acceptor, donor and selected transconjugants.

<p>^a Accession number CP006784</p><p>^b Accession number CP008736</p><p>Plasmids of ESBL242 were isolated, separated and identified by sequencing. The sequences can be accessed at NCBI SRA database: SRX878242. Transconjugants that evolved in different experiments were selected and sequenced.</p><p>Plasmids found in the acceptor, donor and selected transconjugants.</p

Number of evolved transconjugants/ml after co-culturing ESBL242 (donor, amp^R) and <i>E</i>. <i>coli</i> MG1655 (acceptor, chlor^R) for 24 hours in batch culture.

<p>Cells were mixed in a ratio of 1:1 with varying antibiotic concentrations.</p

Number of evolved transconjugants/ml after co-culturing ESBL242 (donor, amp^R) and <i>E</i>. <i>coli</i> MG1655 (acceptor, chlor^R).

<p>Cells were mixed in a ratio of 1:1 at different total cell densities and incubated for 1 hour in batch culture.</p