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, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter 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 cephalosporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cephalosporinase was the only ß-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.

Review of Microfluidic Methods for Cellular Lysis.

Cell lysis is a process in which the outer cell membrane is broken to release intracellular constituents in a way that important information about the DNA or RNA of an organism can be obtained. This article is a thorough review of reported methods for the achievement of effective cellular boundaries disintegration, together with their technological peculiarities and instrumental requirements. The different approaches are summarized in six categories: chemical, mechanical, electrical methods, thermal, laser, and other lysis methods. Based on the results derived from each of the investigated reports, we outline the advantages and disadvantages of those techniques. Although the choice of a suitable method is highly dependent on the particular requirements of the specific scientific problem, we conclude with a concise table where the benefits of every approach are compared, based on criteria such as cost, efficiency, and difficulty.

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 P rnpB , 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.

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.

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.

Empirical model and in vivo characterization of the bacterial response to synthetic gene expression show that ribosome allocation limits growth rate.

Synthetic biology uses modeling to facilitate the design of new genetic constructions. In particular, it is of utmost importance to model the reaction of the cellular chassis when expressing heterologous systems. We constructed a mathematical model for the response of a bacterial cell chassis under heterologous expression. For this, we relied on previous characterization of the growth-rate dependence on cellular resource availability (in this case, DNA and RNA polymerases and ribosomes). Accordingly, we estimated the maximum capacities of the cell for heterologous expression to be 46% of the total RNA and the 33% of the total protein. To experimentally validate our model, we engineered two genetic constructions that involved the constitutive expression of a fluorescent reporter in a vector with a tunable origin of replication. We performed fluorescent measurements using population and single-cell fluorescent measurements. Our model predicted cell growth for several heterologous constructions under five different culture conditions and various plasmid copy numbers with significant accuracy, and confirmed that ribosomes act as the limiting resource. Our study also confirmed that the bacterial response to synthetic gene expression could be understood in terms of the requirement for cellular resources and could be predicted from relevant cellular parameters.

Modular model-based design for heterologous bioproduction in bacteria.

We review the current status of expression of heterologous systems for bioenergy and bioproduction in bacteria using a model-based approach. As an aim for synthetic biology, it requires mathematical models of genetic modules that could be characterized independently of their context. This fastens the design of metabolic circuits using a combinatorial design approach, where given pathways could be optimized for maximal bioproduction, while being nontoxic for the chassis. We show how recent characterization of genetic parts, such as promoters, RBS or sRNAs could be used to fine-tune the expression of individual genes to achieve that goal. We also present lists of enzymes that are used for bioproduction, enlarging such set of biological parts.