Urinary tract infections are one of the most frequent bacterial diseases worldwide. UPECs are the most prominent group of bacterial strains among pathogens responsible for prompting such infections. As a group, these extra-intestinal infection-causing bacteria have developed specific features that allow them to sustain and develop in their inhabited niche of the urinary tract. In this study, we examined 118 UPEC isolates to determine their genetic background and antibiotic resistance. Moreover, we investigated correlations of these characteristics with the ability to form biofilm and to induce a general stress response. We showed that this strain collection expressed unique UPEC attributes, with the highest representation of FimH, SitA, Aer, and Sfa factors (100%, 92.5%, 75%, and 70%, respectively). According to CRA (Congo red agar) analysis, the strains particularly predisposed to biofilm formation represented 32.5% of the isolates. Those biofilm forming strains presented a significant ability to accumulate multi-resistance traits. Most notably, these strains presented a puzzling metabolic phenotype—they showed elevated basal levels of (p)ppGpp in the planktonic phase and simultaneously exhibited a shorter generation time when compared to non-biofilm-forming strains. Moreover, our virulence analysis showed these phenotypes to be crucial for the development of severe infections in the Galleria mellonella model.
Bacteriophage-based applications have a renaissance today, increasingly marking their use in industry, medicine, food processing, biotechnology, and more. However, phages are considered resistant to various harsh environmental conditions; besides, they are characterized by high intra-group variability. Phage-related contaminations may therefore pose new challenges in the future due to the wider use of phages in industry and health care. Therefore, in this review, we summarize the current knowledge of bacteriophage disinfection methods, as well as highlight new technologies and approaches. We discuss the need for systematic solutions to improve bacteriophage control, taking into account their structural and environmental diversity.
Molecular cloning is a routine yet essential technique. Its efficiency relies on ligation and can be greatly improved when using a procedure known as Golden Gate Assembly (GGA). Essential to GGA are type IIS enzymes that have the unique property to cleave downstream their recognition sequence and thus generate any non-palindromic overhangs. Today, GGA benefits from new engineered enzymes with enhanced activity. Concomitantly, high throughput GAG assays (involving the simultaneous study of all overhangs at a time) have proposed optimal GGA substrates with high efficiencies and fidelities. Surprisingly, those assays show either no or unexpected correlation between ligation efficiencies and overhang stabilities. To explain those observations, we present here experiments involving one or two substrates (overhangs) only. When performing GGA at a stable temperature of 37°C, we found that GGA efficiency strongly correlates with overhang stability. Combining those experimental results with a kinetic model, we were able to determine how relevant parameters (time, temperature, molarity, stoichiometry, stacking energy) influence GGA. This work provides a comprehensive view of low-assembly GGA, defines the required optimal conditions and substrates (allowing the fabrication of constructs for single-molecule experiments with unprecedented yields) and gives new insights into DNA ligation that are crucial in biology.
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