An inventory of 44 qPCR assays using hydrolysis probes operating with a unique amplification condition for the detection and quantification of antibiotic resistance genes.

Early antibiotic resistance determinants (ARDs) detection in humans or animals is crucial to counteract their propagation. The ARDs quantification is fundamental to understand the perturbation caused by disruptors, such as antibiotics, during therapies. Forty-three qPCRs on the most diffused ARDs and integrons among human and animal Enterobacterales, and one on the 16S rDNA for bacteria quantification, were developed. The qPCRs, using hydrolysis probes, operated with a unique amplification condition and were tested analytically and diagnostically performing 435 reactions on five positive and negative controls for each qPCR. Diagnostic sensitivity and specificity were confirmed by PCR and genome sequencing of control isolates, demonstrating 100% performance for all qPCRs. An easy and rapid discrimination method for the epidemiologically relevant blaCTX-Ms is provided. This large, noncommercial qPCRs inventory could serve for precise quantification of ARDs, but also as a rapid screening tool for surveillance purposes, providing the basis for further high-throughput developments.

Impact of Antibiotic Therapies on Resistance Genes Dynamic and Composition of the Animal Gut Microbiota.

Antibiotics are major disruptors of the gastrointestinal microbiota, depleting bacterial species beneficial for the host health and favoring the emergence of potential pathogens. Furthermore, the intestine is a reactor of antibiotic resistance emergence, and the presence of antibiotics exacerbates the selection of resistant bacteria that can disseminate in the environment and propagate to further hosts. We reviewed studies analyzing the effect of antibiotics on the intestinal microbiota and antibiotic resistance conducted on animals, focusing on the main food-producing and companion animals. Irrespective of antibiotic classes and animal hosts, therapeutic dosage decreased species diversity and richness favoring the bloom of potential enteropathogens and the selection of antibiotic resistance. These negative effects of antibiotic therapies seem ineluctable but often were mitigated when an antibiotic was administered by parenteral route. Sub-therapeutic dosages caused the augmentation of taxa involved in sugar metabolism, suggesting a link with weight gain. This result should not be interpreted positively, considering that parallel information on antibiotic resistance selection was rarely reported and selection of antibiotic resistance is known to occur also at low antibiotic concentration. However, studies on the effect of antibiotics as growth promoters put the basis for understanding the gut microbiota composition and function in this situation. This knowledge could inspire alternative strategies to antibiotics, such as probiotics, for improving animal performance. This review encompasses the analysis of the main animal hosts and all antibiotic classes, and highlights the future challenges and gaps of knowledge that should be filled. Further studies are necessary for elucidating pharmacodynamics in animals in order to improve therapy duration, antibiotic dosages, and administration routes for mitigating negative effects of antibiotic therapies. Furthermore, this review highlights that studies on aminoglycosides are almost inexistent, and they should be increased, considering that aminoglycosides are the first most commonly used antibiotic family in companion animals. Harmonization of experimental procedures is necessary in this research field. In fact, current studies are based on different experimental set-up varying for antibiotic dosage, regimen, administration, and downstream microbiota analysis. In the future, shotgun metagenomics coupled with long-reads sequencing should become a standard experimental approach enabling to gather comprehensive knowledge on GIM in terms of composition and taxonomic functions, and of ARGs. Decorticating GIM in animals will unveil revolutionary strategies for medication and improvement of animals' health status, with positive consequences on global health.

Mito-priming as a method to engineer Bcl-2 addiction.

Most apoptotic stimuli require mitochondrial outer membrane permeabilization (MOMP) in order to execute cell death. As such, MOMP is subject to tight control by Bcl-2 family proteins. We have developed a powerful new technique to investigate Bcl-2-mediated regulation of MOMP. This method, called mito-priming, uses co-expression of pro- and anti-apoptotic Bcl-2 proteins to engineer Bcl-2 addiction. On addition of Bcl-2 targeting BH3 mimetics, mito-primed cells undergo apoptosis in a rapid and synchronous manner. Using this method we have comprehensively surveyed the efficacy of BH3 mimetic compounds, identifying potent and specific MCL-1 inhibitors. Furthermore, by combining different pro- and anti-apoptotic Bcl-2 pairings together with CRISPR/Cas9-based genome editing, we find that tBID and PUMA can preferentially kill in a BAK-dependent manner. In summary, mito-priming represents a facile and robust means to trigger mitochondrial apoptosis.