First detection of a colistin-resistant Klebsiella aerogenes isolate from a critically ill patient with septic shock in Bulgaria

Colistin is considered as the last-line antibiotic for the treatment of infections caused by extensively drug-resistant Gram-negative pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylo-coccus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enter-obacter species) group. The present study aimed to explore the colistin resistance mechanisms of a Klebsiella aerogenes (formerly Enterobacter aerogenes) isolate (Kae1177-1bg) obtained from a Bulgarian critically ill patient with septic shock in 2020. Antimicrobial susceptibility testing and whole-genome sequencing using DNA nanoball technology were performed. The resulting read pairs were used for draft genome assembly, MLST analysis and mutation screening in the pmrA/B, phoP/Q, and mgrB genes. Kae1177-1bg demonstrated high-level resistance to colistin, resistance to 3rd generation cepha-losporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cepha-losporinase was the only beta-lactamase identified. No mobile colistin resistance (mcr) genes were detected. A total of three missense variants in the genes for the two-component PmrA/PmrB system were identified. Two of them were located in the pmrB (pR57K and pN275K) and one in the pmrA gene (pL162M). The pN275K variant emerged as the most likely cause for colistin resistance because it affected a highly conservative position and was the only nonconservative amino acid substitution. In conclusion, to the best of our knowledge, this is the first documented clinical case of a high-level colistin-resistant K. aerogenes in Bulgaria and the first identification of the nonconservative amino acid substitution pN275K worldwide. Colistin-resistant Gram-negative pathogens of ESKAPE group are serious threat to public health and should be subjected to infection control stewardship practices

Expanding the toolbox for Synechocystis sp. PCC 6803 : validation of replicative vectors and characterization of a novel set of promoters

Cyanobacteria are promising ‘low-cost’ cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the ‘green’ Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference PrnpB, three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study

Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression

[EN] Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.EVOPROG [FP7-ICT-610730]; PROMYS [FP7-KBBE-613745 to A.J.]; Ministerio de Economia y Competitividad, Spain [BIO2011-26741 to J.-A.D.]; PRES Paris Sud grant (S.S.); EMBO long-term fellowship co-funded by Marie Curie actions [ALTF-1177-2011 A.J., G.R.]; AXA research fund; Ministerio de Educacion, Cultura y Deporte, Spain [AP2012-3751 to E.M.]. Funding for open access charge: EVOPROG [FP7-ICT-610730]; PROMYS [FP7-KBBE-613745].Shen, S.; Rodrigo Tarrega, G.; Prakash, S.; Majer, E.; Landrain, T.; Kirov, B.; Daros Arnau, JA.... (2015). Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression. Nucleic Acids Research. 43(10):5158-5170. https://doi.org/10.1093/nar/gkv287S515851704310Ulrich, L. E., Koonin, E. V., & Zhulin, I. B. (2005). One-component systems dominate signal transduction in prokaryotes. 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Mapping genomic loci implicates genes and synaptic biology in schizophrenia

Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Conception, construction et caractérisation de circuits génétiques dynamiques dans des bactéries

La conception et la construction de "parts" en biologie synthétique n'est pas triviale et nécessite de nombreuses conditions. Les "parts" utilisées dans des circuits génétiques devraient être modulaires, bien caractérisées avec un devenir précis et robustes aux changements de l'environnement. Elles devraient être résistantes aux interférences avec l'environnement et aux mutations. Aussi, elles devraient être proprement modélisées sur la base de paramètres dérivés d'expériences au niveau d'une seule cellule. Dans ma thèse, j'ai cherché en détails les conditions nécessaires à l'ingénierie de "parts" individuelles telles que promoteurs, sites de fixation de ribosomes, facteurs de transcription et quelques importants types de dispositifs. De plus, j'ai établi une plate-Forme pour la caractérisation de dispositifs génétiques au niveau d'une cellule unique. Tout le matériel et savoir-Faire nécessaires à l'ingénierie de dispositifs microfluidiques ont été acquis. Le procédé complet depuis la conception de dispositifs microfluidiques de leur fabrication à leur utilisation fonctionnelle pour des expériences microbiennes a été développée avec succès. Un outil d'analyse d'images acquises à partir d'expériences de microscopie parallélisé sur ordinateur a aussi été developpé. Les résultats expérimentaux ont prouvé que les dispositifs développés avaient un comportement conforme aux attentes théoriques. De plus, les protocoles expérimentaux, de fabrication et l'analyse automatique de données se sont avérés être adaptés et efficaces pour la caractérisation au niveau d'une cellule unique des bactéries développées.The task to design and construct parts for the synthetic biology is not simple and needs to meet a number of requirements. The parts utilized for the construction of genetic circuits should be modular, well-Characterized, well-Behaved and robust to changes in the environment. They should be insulated from cross-Talk with the environment and be resilient to mutations. Finally, they should also be properly modeled based on parameters derived from single-Cell level experiments. In my thesis, i researched in detail the general requirements for the engineering of individual parts like promoters, ribosome binding site, transcription factors and of some important type of devices. Furthermore, i established a complete platform for the single-Cell level characterization of engineered genetic devices. All the required hardware and know-How for the fabrication of microfluidics devices capable of sustained bacterial growth was acquired. The whole process from the design of microfluidics devices that aimed functionality to their fabrication and utilization for microbial experiments was successfully developed. An efficient image-Processing tool for distributed computational analysis of the data acquired during the microscopy experiments was also developed. The experimental results proved that the engineered genetic devices were behaving according to theoretical expectations. Furthermore, the established experimental procedures, fabrication process and automated data analysis showed to be well-Adapted to the task of single-Cell characterization of engineered bacteria and efficient

An automated approach for single-cell tracking in epifluorescence microscopy applied to E. coli growth analysis on microfluidics biochips

International audienceWith the accumulation of knowledge for the intimate molecular mechanisms governing the processes inside the living cells in the later years, the ability to characterize the performance of elementary genetic circuits and parts at the single-cell level is becoming of crucial importance. Biological science is arriving to the point where it can develop hypothesis for the action of each molecule participating in the biochemical reactions and need proper techniques to test those hypothesis. Microfluidics is emerging as the technology that combined with high-magnification microscopy will allow for the long-term single-cell level observation of bacterial physiology. In this study we design, build and characterize the gene dynamics of genetic circuits as one of the basic parts governing programmed cell behavior. We use E. coli as model organism and grow it in microfluidics chips, which we observe with epifluorescence microscopy. One of the most invaluable segments of this technology is the consequent image processing, since it allows for the automated analysis of vast amount of single-cell observation and the fast and easy derivation of conclusions based on that data. Specifically, we are interested in promoter activity as function of time. We expect it to be oscillatory and for that we use GFP (green fluorescent protein) as a reporter in our genetic circuits. In this paper, an automated framework for single-cell tracking in phase-contrast microscopy is developed, combining 2D segmentation of cell time frames and graph-based reconstruction of their spatiotemporal evolution with fast tracking of the associated fluorescence signal. The results obtained on the investigated biological database are presented and discussed

Novel Microfluidics Device for Rapid Antibiotics Susceptibility Screening

In recent years, excessive utilization of antibiotics has led to the emergence of antibiotic microbial resistance on a planetary scale. This recent phenomenon represents a serious threat to public health, as well as an enormous burden for healthcare systems’ budgets worldwide. Novel, rapid and cheap methods for antibiotic susceptibility screening are urgently needed for this obstacle to be overcome. In this paper, we present a microfluidic device for on-chip antibiotic resistance testing, which allows for antibiotic microbial resistance detection within 6 hours. The design, fabrication and experimental utilization of the device are thoroughly described and analyzed, as well as possibilities for future automation of the whole process. The accessibility of such a device for all people, regardless of economic status, was of utmost importance for us during the development of the project

Theoretical and experimental analysis of the forced LacI-AraC oscillator with a minimal gene regulatory model

Oscillatory dynamics have been observed in multiple cellular functions and synthetic constructs; and here, we study the behavior of a synthetic oscillator under temporal perturbations. We use a minimal model, involving a single transcription factor with delayed self-repression and enzymatic degradation, together with a first-order perturbative approach, to derive an analytical expression for the power spectrum of the system, which characterizes its response to external forces and molecular noise. Experimentally, we force and monitor the dynamics of the LacI-AraC oscillator in single cells during long time intervals by constructing a microfluidics device. Pulse dynamics of IPTG with different periods serve to perturb this system. Due to the resonance of the system, we predict theoretically and confirm experimentally the dependence on the forcing frequency of the variability in gene expression with time and the synchronization of the population to the input signal. The reported results show that the engineering of gene circuits can provide test cases for dynamical models, which could be further exploited in synthetic biology