Trayectorias evolutivas de las mutaciones que confieren resistencia a gentamicina en Escherichia coli

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 16-10-201

Detection and Control of Indoor Airborne Pathogenic Bacteria by Biosensors Based on Quorum Sensing Chemical Language: Bio-Tools, Connectivity Apps and Intelligent Buildings

Nowadays, lifestyles and climate change lead people to spend long periods in indoors spaces, where reduced ventilation and artificial light favor the concentration and spread of airborne pathogenic microorganisms. Current procedures for microbiological air evaluation are based on air sampling coupled to traditional microbiological culture-dependent methods such as biochemical tests and molecular rDNA 16S sequencing. These techniques generate an important delay in the application of prevention and control measures. This chapter presents whole cell-based biosensors that are able to detect quorum sensing signaling molecules produced by airborne pathogenic bacteria as a tool for indoor air monitoring. Furthermore, a general biosensor model is proposed. In this model, in vivo biosensors technology can be connected to online applications (Apps), being part of intelligent buildings, in order to reduce airborne pathogenic bacteria concentration and dissemination

Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates

Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE, and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci

Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates

Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE, and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci

DataSheet1.docx

<p>Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE, and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci.</p

A whole‐cell hypersensitive biosensor for beta‐lactams based on the AmpR‐AmpC regulatory circuit from the Antarctic Pseudomonas sp. IB20

Abstract Detecting antibiotic residues is vital to minimize their impact. Yet, existing methods are complex and costly. Biosensors offer an alternative. While many biosensors detect various antibiotics, specific ones for beta‐lactams are lacking. To address this gap, a biosensor based on the AmpC beta‐lactamase regulation system (ampR–ampC) from Pseudomonas sp. IB20, an Antarctic isolate, was developed in this study. The AmpR–AmpC system is well‐conserved in the genus Pseudomonas and has been extensively studied for its involvement in peptidoglycan recycling and beta‐lactam resistance. To create the biosensor, the ampC coding sequence was replaced with the mCherry fluorescent protein as a reporter, resulting in a transcriptional fusion. This construct was then inserted into Escherichia coli SN0301, a beta‐lactam hypersensitive strain, generating a whole‐cell biosensor. The biosensor demonstrated dose‐dependent detection of penicillins, cephalosporins and carbapenems. However, the most interesting aspect of this work is the high sensitivity presented by the biosensor in the detection of carbapenems, as it was able to detect 8 pg/mL of meropenem and 40 pg/mL of imipenem and reach levels of 1–10 ng/mL for penicillins and cephalosporins. This makes the biosensor a powerful tool for the detection of beta‐lactam antibiotics, specifically carbapenems, in different matrices

Design, synthesis and biological evaluation of non-covalent AmpC Beta-Lactamases inhibitors

Bacterial resistance represents a worldwide emergency threatening the efficacy of all available antibiotics. Among the several resistance mechanisms developed by bacteria, beta-lactamases enzymes (BLs), able to inactivate most beta-lactam core antibiotics, represent a key target to block prolonging antibiotics half-life. Several approaches aimed at inhibiting BLs have been so far undertaken, mainly involving beta-lactam like or covalent inhibitors. Applying a structure based de novo design approach, we recently discovered a novel, non-covalent and competitive inhibitor of AmpC beta lactamases: it has a Ki of 1 µM, a ligand efficiency of 0.38 kcal ∙ mol–1 and lead-like physical properties. Moreover, it reverts resistance to ceftazidime in bacterial pathogens expressing AmpC and does not up-regulate beta-lactamase expression in cell culture. Its features make it a good candidate for chemical optimization: starting from its crystallographic complex with AmpC, eleven analogues were designed to complement additional AmpC sites, then synthesized and tested against clinical resistant pathogens. While the new inhibitors maintain similar in vitro activity as the starting lead, some of them exert a higher potency in in vivo tests, showing improved synergic potency with ceftazidime in resistant clinically isolated strains

Computational and biological profile of boronic acids for the detection of bacterial serine- and metallo-β-lactamases

β-Lactamases (BLs) able to hydrolyze β-lactam antibiotics and more importantly the last resort carbapenems, represent a major mechanism of resistance in Gram-negative bacteria showing multi-drug or extensively drug resistant phenotypes. The early detection of BLs responsible of resistant infections is challenging: approaches aiming at the identification of new BLs inhibitors (BLI) can thus serve as the basis for the development of highly needed diagnostic tools. Starting from benzo-[b]-thiophene-2-boronic acid (BZB), a nanomolar inhibitor of AmpC β-lactamase (K i  = 27 nM), we have identified and characterized a set of BZB analogues able to inhibit clinically-relevant β-lactamases, including AmpC, Extended-Spectrum BLs (ESBL), KPC- and OXA-type carbapenemases and metallo-β-lactamases (MBL). A multiligand set of boronic acid (BA) β-lactamase inhibitors was obtained using covalent molecular modeling, synthetic chemistry, enzyme kinetics and antibacterial susceptibility testing. Data confirmed the possibility to discriminate between clinically-relevant β-lactamases on the basis of their inhibition profile. Interestingly, this work also allowed the identification of potent KPC-2 and NDM-1 inhibitors able to potentiate the activity of cefotaxime (CTX) and ceftazidime (CAZ) against resistant clinical isolates (MIC reduction, 32-fold). Our results open the way to the potential use of our set of compounds as a diagnostic tool for the sensitive detection of clinically-relevant β-lactamases.This work was supported by the OPTObacteria project within the 7FP (Grant agreement no: 286998; www.optobacteria.eu), and by the National Institute of Health (Grant no: GM63815). JB was supported by the Spanish Network for Research in Infectious Pathology (REIPI) RD12/0015/0012, funded by Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, and co-financed by the European Development Regional Fund “A way to achieve Europe” ERDF.Peer reviewe

Table_1_Commonalities between the Atacama Desert and Antarctica rhizosphere microbial communities.DOCX

Plant-microbiota interactions have significant effects on plant growth, health, and productivity. Rhizosphere microorganisms are involved in processes that promote physiological responses to biotic and abiotic stresses in plants. In recent years, the interest in microorganisms to improve plant productivity has increased, mainly aiming to find promising strains to overcome the impact of climate change on crops. In this work, we hypothesize that given the desertic environment of the Antarctic and the Atacama Desert, different plant species inhabiting these areas might share microbial taxa with functions associated with desiccation and drought stress tolerance. Therefore, in this study, we described and compared the composition of the rhizobacterial community associated with Deschampsia antarctica (Da), Colobanthus quitensis (Cq) from Antarctic territories, and Croton chilensis (Cc), Eulychnia iquiquensis (Ei) and Nicotiana solanifolia (Ns) from coastal Atacama Desert environments by using 16S rRNA amplicon sequencing. In addition, we evaluated the putative functions of that rhizobacterial community that are likely involved in nutrient acquisition and stress tolerance of these plants. Even though each plant microbial rhizosphere presents a unique taxonomic pattern of 3,019 different sequences, the distribution at the genus level showed a core microbiome with a higher abundance of Haliangium, Bryobacter, Bacillus, MND1 from the Nitrosomonadaceae family, and unclassified taxa from Gemmatiamonadaceae and Chitinophagaceae families in the rhizosphere of all samples analyzed (781 unique sequences). In addition, species Gemmatirosa kalamazoonesis and Solibacter usitatus were shared by the core microbiome of both Antarctic and Desert plants. All the taxa mentioned above had been previously associated with beneficial effects in plants. Also, this microbial core composition converged with the functional prediction related to survival under harsh conditions, including chemoheterotrophy, ureolysis, phototrophy, nitrogen fixation, and chitinolysis. Therefore, this study provides relevant information for the exploration of rhizospheric microorganisms from plants in extreme conditions of the Atacama Desert and Antarctic as promising plant growth-promoting rhizobacteria.</p