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The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real- time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10−1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase- producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M- 15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

The synthesis, photophysical, and electrochemical properties of selectively mono-, bis- and tris-dimethylamino- and trimethylammonium-substituted bis-triarylborane bithiophene chromophores are presented along with the water solubility and singlet oxygen sensitizing efficiency of the cationic compounds Cat1+ , Cat2+ , Cat(i)2+ , and Cat3+ . Comparison with the mono-triarylboranes reveals the large influence of the bridging unit on the properties of the bis-triarylboranes, especially those of the cationic compounds. Based on these preliminary investigations, the interactions of Cat1+ , Cat2+ , Cat(i)2+ , and Cat3+ with DNA, RNA, and DNApore were investigated in buffered solutions. The same compounds were investigated for their ability to enter and localize within organelles of human lung carcinoma (A549) and normal lung (WI38) cells showing that not only the number of charges but also their distribution over the chromophore influences interactions and staining properties.

K. pneumoniae isolates often harbor various antibiotic resistance determinants including extended-spectrum β-lactamases (ESBLs), plasmid- mediated AmpC β-lactamases (p-Amp-C) and carbapenemases. In this study we analyzed 65 K. pneumoniae isolates obtained from urinary tract infections in the outpatients setting, with regard to antibiotic susceptibility, β-lactamase production, virulence traits and plasmid content. Antibiotic susceptibility was determined by broth microdilution method. PCR was applied to detect genes encoding ESBLs, p-Amp-C and carbapenemases and plasmid incompatibility groups. Phenotypic methods were applied to characterize virulence determinants. Increasing resistance trend was observed for amoxicillin/clavulanate, imipenem, meropenem and ciprofloxacin. The study showed that ESBLs belonging to the CTX-M family, conferring high level of resistance to expanded-spectrum cephalosporins (ESC) were the dominant resistance trait among early isolates (2013 to 2016) whereas OXA-48 carbapenemase, belonging to class D, emerged in significant numbers after 2017. OXA-48 producing organisms coharbored ESBLs. KPC-2 was dominant among isolates from Dubrovnik in the recent years. Colistin resistance was reported in three isolates. Inc L/M was the dominant plasmid in the later period, encoding OXA- 48. Hyperviscosity was linked to KPC positivity and emerged in the later period. This report describes evolution of antibiotic resistance in K. pneumoniae from ESBLs to carbapenemases and colistin resistance. The study demonstrated the ability of K. pneumoniae to acquire various resistance determinants, over time. The striking diversity of the UTI isolates could result from introduction of the isolates from the hospitals, transfer of plasmids and multidirectional evolution.

The growth of surface-attached single-stranded deoxyribonucleic acid (ssDNA) chains is monitored in situ using an evanescent wave optical biosensor that combines surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The "grafting-from" growth of ssDNA chains is facilitated by rolling circle amplification (RCA), and the gradual prolongation of ssDNA chains anchored to a gold sensor surface is optically tracked in time. At a sufficient density of the polymer chains, the ssDNA takes on a brush architecture with a thickness exceeding 10 μm, supporting a spectrum of guided optical waves traveling along the metallic sensor surface. The simultaneous probing of this interface with the confined optical field of surface plasmons and additional more delocalized dielectric optical waveguide modes enables accurate in situ measurement of the ssDNA brush thickness, polymer volume content, and density gradients. We report for the first time on the utilization of the SPR/OWS technique for the measurement of the RCA speed on a solid surface that can be compared to that in bulk solutions. In addition, the control of ssDNA brush properties by changing the grafting density and ionic strength and post-modification via affinity reaction with complementary short ssDNA staples is discussed. These observations may provide important leads for tailoring RCA toward sensitive and rapid assays in affinity-based biosensors.

A DNA aptazyme consists of an aptamer domain and a DNAzyme module, in which the DNAzyme activity can be regulated by the aptamer-target interaction. The complex of G-quadruplex (GQ) and hemin is a peroxidase-mimicking DNAzyme and has become increasingly popular as a reporter system for biosensing applications. The development of GQ-based aptazymes is of high interest as they can be used as label-free biosensors for the real-time detection of pathogens. Herein, we rationally designed ca. 200 GQ-based aptazyme candidates and evaluated the suitability of 14 aptamers targeting quinine, Protein A, Staphylococcus enterotoxin B, and ATP for this detection concept. As a result, six novel aptazymes were developed for the specific detection of quinine based on two quinine-binding aptamers. The rest of designed probes, however, hardly showed significant functionality. To uncover the reasons, we performed enzyme-linked oligonucleotide assays to find how the affinity of aptamers is affected once conjugated to the DNAzyme sequence or upon integration into the aptazyme probe. Furthermore, we investigated the impact of the structure-switching functionality in the parent aptamer and the effect of the reaction matrix on the efficiency of probes.

The effect of different branching types of glycosylation on the structure and dynamics of the horseradish peroxidase (HRP) and an engineered split horseradish peroxidase (sHRP) was studied using all-atom molecular dynamics (MD) simulations. Although tertiary structures of both proteins are stable in the presence, as well as in the absence of glycans, differences in the dynamical properties regarding the presence of glycans were noticed. Fluctuations in the protein structure along both proteins are decreased when glycosylation is introduced. We identified two main regions that are affected the most. The peripheral region is impacted directly by glycans and the central region within the active site with a propagated effect of glycans. Since the mentioned central region in the glycoprotein is not surrounded by glycans and is close to the heme, it is easily approachable to the solvent and substrate. An influence of the glycan presence on the electrostatic potential of the protein and on the heme cofactor was also observed. Altogether, this work presents a global and local analysis of the glycosylation influence on HRP protein's structural and dynamical properties at a molecular level.

Mechanical forces produced by motor proteins and microtubule dynamics within the mitotic spindle are crucial for the movement of chromosomes and their segregation into the daughter cells. In addition to linear forces, rotational forces or torques are present in the spindle, reflected in the left-handed twisted shapes of microtubule bundles that make the spindle chiral. However, the biological role and molecular origins of spindle chirality are unknown. By developing methods to measure spindle twist, we show that spindles are most chiral near the metaphase-to-anaphase transition. To test whether spindles react to external forces by changing the twist, we compressed the spindles along their axis, which resulted in stronger left-handed twist. Inhibition or depletion of motor proteins that perform chiral stepping, Eg5/kinesin-5 or Kif18A/kinesin-8, decreased the left-handed twist or led to right-handed twist, suggesting that these motors regulate the twist by rotating microtubules around one another within the antiparallel overlaps. Right-handed twist was also observed after depletion of the microtubule crosslinker PRC1 or the nucleator augmin, indicating that PRC1 contributes to the twist by constraining microtubule rotation, and augmin by nucleating antiparallel bridging microtubules. The observed switch from left-handed to right-handed twist reveals the existence of competing mechanisms that promote twisting in opposite directions. As round spindles were more twisted than elongated ones, we suggest that bending and twisting moments are generated by similar molecular mechanisms. We propose a physiological role for spindle chirality in providing a passive mechanical response to forces, decreasing the risk of spindle breakage under high load.

Bloodstream infections (BSI) are life-threatening conditions. Inappropriate initial antimicrobial therapy leads to higher mortality in patients with septic shock [1]. Infections due to multidrug resistant (MDR) pathogens are prone to treatment failures and inappropriate empiric antimicrobial therapy, but whether drug resistance alone increases mortality in the setting of appropriate therapy is unclear. Bacterial resistance to antibiotics is growing up day by day, in both community and hospital setting, increasing morbidity and mortality. Therapy of invasive infections due to MDR Gram-negative bacteria is challenging, and some of the few active drugs are not available in many countries. Global spread of multiresistant pathogens and the lack of new antibiotics are limiting clinicians in the therapy of septicemia. Particularly important are ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species [2, 3]. Therapeutic failures are usually associated with methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), extended-spectrum β- lactamase (ESBL) producing Enterobacterales, carbapenem-resistant Enterobacterales (CRE), carbapenem resistant Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB) [3]. Risk factors for acquiring bloodstream infections with MDR bacteria are: frequent antibiotic treatments, indwelling urinary and intravascular catheters, recent surgical procedures and stay in the hospital ICU. ESBLs hydrolyse expanded- spectrum cephalosporins (ESC) and render them inactive. The spread of plasmids carrying CTX-M type genes in the community beginning mostly in the 2000s is the main driver of ESBL dissemination in infections associated with Enterobacterales, including BSI isolates and replaced other ESBL enzymes (TEM and SHV) that were identified in hospital infections in the past [4, 5]. Previous studies revealed very high rates of ESBL producing Enterobacteriaceae associated with BSI in South America, the Far East and Mediterranean countries [6–8]. The rate of ESBLs in the previous studies ranged from 3% to 15%. CTX-M-15 ESBL was found in E. coli causing bloodstream infections in Croatia [9]. The isolates exhibited resistance to expanded-spectrum cephalosporins (ESC), gentamicin and ciprofloxacin. CTX-M- ESBLs are also dominant in South America [6, 7], but the SHV and TEM type are still present in some geographic areas such as the Far East [8]. In the previous studies the clinical outcome did not differ between patients with ESBL- positive and ESBL- negative infections. However, the prior usage of antibiotics and inappropriate empirical therapy was more frequent among patients with ESBL- positive organisms [8]. Haematologic malignancies and recent chemotherapy were more frequent in the ESBL- positive group (35% vs 8, 3%) and (54% vs 34%) respectively. A high level of resistance to ESC, gentamicin, and ciprofloxacin was noticed. Except for ESBLs, multidrug- resistant AmpC- producing organisms such as Enterobacter spp, Serratia spp and Citrobacter spp can also cause septicaemia and are challenging for treatment [10]. Carbapenems are usually the antibiotics of choice for the treatment of BSI due to ESBL- or AmpC- producing organisms. Recently, carbapenem-resistant isolates, associated with carbapenemases belonging to class A, B or D, were reported to be associated with BSI, particularly in developing countries [11, 12], contributing to the poor outcome. Carbapenem resistance in P. aeruginosa was shown to cause increased mortality in BSI [13]. In this study, blood samples were collected alongside with routine blood cultures (BC) from the patients with suspected sepsis, to evaluate the prevalence of different causative agents/positive BCs in patients with suspected bacteraemia, taken in the frames of the European FAPIC project (Fast assay for pathogen identification and characterization, Horizon, 2020).

The synthesis, photophysical, and electrochemical properties of selectively mono‐, bis‐ and tris‐dimethylamino‐ and trimethylammonium‐substituted bis‐triarylborane bithiophene chromophores are presented along with the water solubility and singlet oxygen sensitizing efficiency of the cationic compounds Cat1+ , Cat2+ , Cat(i)2+ , and Cat3+ . Comparison with the mono‐triarylboranes reveals the large influence of the bridging unit on the properties of the bis‐triarylboranes, especially those of the cationic compounds. Based on these preliminary investigations, the interactions of Cat1+ , Cat2+ , Cat(i)2+ , and Cat3+ with DNA, RNA, and DNApore were investigated in buffered solutions. The same compounds were investigated for their ability to enter and localize within organelles of human lung carcinoma (A549) and normal lung (WI38) cells showing that not only the number of charges but also their distribution over the chromophore influences interactions and staining properties., Four selectively mono ‐, bis ‐ and tris ‐trimethylammonium‐substituted, bithiophene‐bridged bis‐triarylboranes were synthesized and their potential biological applications were investigated including live cell imaging and photo‐induced cell death. It is demonstrated that their photophysical and electrochemical properties and their interactions with DNAs, RNA and cells, are strongly influenced by the number and distribution of charges within the compounds.

The exposure of humans to nano-and microplastic particles (NMPs) is an issue recognized as a potential health hazard by scientists, authorities, politics, non-governmental organizations and the general public. The concentration of NMPs in the environment is increasing concomitantly with global plastic production and the usage of plastic materials. NMPs are detectable in numerous aquatic organisms and also in human samples, therefore necessitating a risk assessment of NMPs for human health. So far, a comprehensive risk assessment of NMPs is hampered by limited availability of appropriate reference materials, analytical obstacles and a lack of definitions and standardized study designs. Most studies conducted so far used polystyrene (PS) spheres as a matter of availability, although this polymer type accounts for only about 7% of total plastic production. Differently sized particles, different concentration and incubation times, and various biological models have been used, yielding hardly comparable data sets. Crucial physico-chemical properties of NMPs such as surface (charge, polarity, chemical reactivity), supplemented additives and adsorbed chemicals have been widely excluded from studies, although in particular the surface of NMPs determines the interaction with cellular membranes. In this manuscript we give an overview about the critical parameters which should be considered when performing risk assessments of NMPs, including novel reference materials, taking into account surface modifications (e.g., reflecting weathering processes), and the possible role of NMPs as a substrate and/or carrier for (pathogenic) microbes. Moreover, we make suggestions for biological model systems to evaluate immediate toxicity, long-term effects and the potential of NMPs to cross biological barriers. We are convinced that standardized reference materials and experimental parameters along with technical innovations in (nano)-particle sampling and analytics are a prerequisite for the successful realization of conclusive human health risk assessments of NMPs. ispartof: INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH vol:17 issue:23 ispartof: location:Switzerland status: published

The success of any oligonucleotide-based experiment strongly depends on the accurate design of the components. Oli2go is a user-friendly web tool that provides efficient multiplex oligonucleotide design including specificity and primer dimer checks. Its fully automated workflow involves important design steps that use specific parameters to produce high-quality oligonucleotides. This chapter describes how these steps are computationally implemented by oli2go.

The success of any oligonucleotide-based experiment strongly depends on the accurate design of the components. Oli2go is a user-friendly web tool that provides efficient multiplex oligonucleotide design including specificity and primer dimer checks. Its fully automated workflow involves important design steps that use specific parameters to produce high-quality oligonucleotides. This chapter describes how these steps are computationally implemented by oli2go.

The polymerase chain reaction is not only essential for many DNA-based diagnostic methods but is also exploited in other molecular methods that require an upstream amplification step. Multiplex PCRs are especially attractive as they reduce the number of individual reactions. However, the multiplexing efficiency is impaired by primer interactions such as the formation of primer dimers. In this study, covalent crosslinking of primers via their 5'-ends was used to avoid those undesired effects. The specificity of the primers as well as the efficiency of the PCR could be increased upon primer crosslinking in reactions containing up to 34 primer pairs targeting the most important antibiotic resistance genes in a single multiplex reaction.

DNA nanotechnology is a rapidly advancing field, which increasingly attracts interest in many different disciplines, such as medicine, biotechnology, physics and biocomputing. The increasing complexity of novel applications requires significant computational support for the design, modelling and analysis of DNA nanostructures. However, current in silico design tools have not been developed in view of these new applications and their requirements. Here, we present Adenita, a novel software tool for the modelling of DNA nanostructures in a user-friendly environment. A data model supporting different DNA nanostructure concepts (multilayer DNA origami, wireframe DNA origami, DNA tiles etc.) has been developed allowing the creation of new and the import of existing DNA nanostructures. In addition, the nanostructures can be modified and analysed on-the-fly using an intuitive toolset. The possibility to combine and re-use existing nanostructures as building blocks for the creation of new superstructures, the integration of alternative molecules (e.g. proteins, aptamers) during the design process, and the export option for oxDNA simulations are outstanding features of Adenita, which spearheads a new generation of DNA nanostructure modelling software. We showcase Adenita by re-using a large nanorod to create a new nanostructure through user interactions that employ different editors to modify the original nanorod.

Nanomechanical devices are becoming increasingly popular due to the very diverse field of potential applications, including nanocomputing, robotics, and drug delivery. DNA is one of the most promising building materials to realize complex 3D structures at the nanoscale level. Several mechanical DNA origami structures have already been designed capable of simple operations such as a DNA box with a controllable lid, bipedal walkers, and cargo sorting robots. However, the nanomechanical properties of mechanically interlinked DNA nanostructures that are in general highly deformable have yet to be extensively experimentally evaluated. In this work, a multicomponent DNA origami-based rotor is created and fully characterized by electron microscopy under negative stain and cryo preparations. The nanodevice is further immobilized on a microfluidic chamber and its Brownian and flow-driven rotational behaviors are analyzed in real time by single-molecule fluorescence microscopy. The rotation in previous DNA rotors based either on strand displacement, electric field or Brownian motion. This study is the first to attempt to manipulate the dynamics of an artificial nanodevice with fluidic flow as a natural force.

Background: The prevalence of Escherichia coli strains producing extended-spectrum β-lactamases (ESBLs) has increased both in the community and in healthcare settings. Furthermore, recent studies in nursing homes and long-term care facilities have shown that these institutions can act as potential reservoirs of ESBL- and CTX-M–producing E. coli. Consequently, we aimed to characterize ESBLs produced by E. coli isolates causing hospital-onset, long-term care facility and community infections throughout Croatia (Europe), as well as to compare antimicrobial sensitivity patterns, molecular specificities, plasmid types and epidemiological features. Methods: From a total pool of 16,333 E. coli isolates, 164 ESBL-producing strains with reduced susceptibility to third-generation cephalosporins were used for further appraisal. Phenotypic tests for the detection of ESBLs and plasmid-mediated AmpC β-lactamases were initially pursued (including a novel version of modified CIM test named cephalosporin inactivation method), followed by conjugation experiments, molecular detection of resistance genes, plasmid extraction with PCR-based replicon typing, serotyping, genotyping with pulsed-field gel electrophoresis, and whole-genome sequencing (WGS). Results: The isolates in this study exhibited a high level of resistance to expanded-spectrum cephalosporins and carried CTX-M or TEM β-lactamases, and all of them were classified as multidrug-resistant due to their resistance pattern to other antimicrobial drugs. The β-lactamase content did not differ among isolated E. coli strains from various sources (ie, hospitals, nursing homes, and the community). According to the genotyping results, the isolates were allocated into 8 clusters, which contained subclusters. Serotyping results revealed that O25 antigen was the dominant one; furthermore, isolates subjected to WGS belonged to the ST131 sequence type. The most pervasive plasmid types in the isolates from the country’s capital (Zagreb) were IncFII and FIA, whereas FIA alone was a dominant plasmid type in the southern part of the country. Conversely, eastern parts were characterized by plasmids belonging to IncB/O and IncW groups. Conclusions: Our study demonstrated the dissemination of group 1 CTX-M–positive E. coli not only in different geographic regions of Croatia but also in different arms of the healthcare system (ie, hospitals, nursing homes, and the community). Our results also confirmed the switch from previously pervasive SHV-2 and SHV-5 ESBLs to the nationwide predominance of group 1 CTX-M β-lactamases; however, regional distribution was associated with different plasmid types carrying blaCTX-M genes. These types of nationwide studies are indispensable for informing global decision making that addresses the issue of antimicrobial resistance.Funding: NoneDisclosures: None

Contains fulltext : 229512.pdf (Publisher’s version ) (Open Access) Fast microbiological diagnostics (MDx) are needed to ensure early targeted antimicrobial treatment in sepsis. This systematic review focuses on the impact on antimicrobial management and patient outcomes of MDx for pathogen and resistance gene identification compared with blood cultures. PubMed was searched for clinical studies using either whole blood directly or after short-term incubation. Twenty-five articles were retrieved describing the outcomes of 8 different MDx. Three interventional studies showed a significant increase in appropriateness of antimicrobial therapy and a nonsignificant change in time to appropriate therapy. Impact on mortality was conflicting. Length of stay was significantly lower in 2 studies. A significant decrease in antimicrobial cost was demonstrated in 6 studies. The limitations of this systematic review include the low number and observed heterogeneity of clinical studies. In conclusion, potential benefits of MDx regarding antimicrobial management and some patient outcomes were reported. More rigorous intervention studies are needed focusing on the direct benefits for patients.

In recent years, a dramatic increase in the prevalence of Escherichia coli strains producing extended-spectrum β-lactamases (ESBLs) has been observed - both in the community and in healthcare settings. This multicentric study aimed to characterize ESBLs produced by E. coli isolates causing hospital- onset and community urinary tract infections, as well as to compare their antimicrobial sensitivity patterns, β-lactamase content and plasmid types. Phenotypic tests for the detection of ESBLs and plasmid-mediated AmpC β- lactamases were initially pursued, followed by molecular detection of resistance genes, plasmid characterization, genotyping with pulsed-field gel electrophoresis and whole genome sequencing (WGS). The isolates exhibited high level of resistance to expanded-spectrum cephalosporins (ESC) and carried CTX-M (cefotaximase-Munich) or TEM (Temoniera) β- lactamases. All six representative isolates subjected to WGS belonged to the widespread clone ST131. In conclusion, our study demonstrated dissemination of group 1 CTX-M positive E. coli in different geographic regions of Croatia, but also different components of the health care systems (hospitals, nursing homes and the community) and confirmed the switch from SHV-2 (suphydril variant) and SHV-5 ESBLs to the nation-wide predominance of group 1 CTX-M β-lactamases. Different plasmids were shown to be associated with the dissemination of blaCTX-M genes in different geographic regions of Croatia.

Colistin is a last-resort antibiotic for the treatment of infections caused by multidrug and carbapenem-resistant Gram-negative bacteria. Colistin resistance has been emerging and multiple outbreaks have been reported in Europe and elsewhere. It has been most frequently reported in carbapenem-resistant K. pneumoniae. In this study, 24 multidrug and colistin-resistant clinical isolates (14 K. pneumoniae, one E. aerogenes, one E. cloacae, and eight A. baumannii) were collected from four hospitals in Croatia from 2013 to 2018, in order to analyse the molecular epidemiology and mechanisms of antibiotic resistance. beta-lactamase and carbapenemase genes were detected by PCR. Genotyping was done on selected isolates by rep-PCR. Whole genome sequencing (WGS) was performed to discover possible molecular mechanisms for the observed colistin resistance. All isolates, except two K. pneumoniae isolates, were extensively drug resistant. Ten out of 16 (63%) K. pneumoniae isolates possessed bla(OXA-48), which is the most common carbapenem resistance gene in Croatia and in other parts of Europe. All A. baumannii isolates possessed the OXA-23-like carbapenem hydrolysing oxacillinase and five turned out to be pandrug-resistant. Colistin resistance was most likely chromosomally mediated. After sequence analysis, none of the isolates were found to possess any of the mcr gene variants. Several previously reported mutations were found in PmrB, PhoP, PhoQ, and MgrB, which are associated with colistin resistance. In the global phylogenetic analysis, DNA mutations causing mutations in the MgrB protein were present mostly in lineages comprising colistin resistant isolates, and the second most prevalent mutation (K3X) was also encountered in our isolates. In addition, based on genotyping by rep-PCR, the spread of colistin resistance is most likely to be clonal. Most importantly, the presence of colistin resistance together with carbapenemase genes in extensively drug resistant isolates poses real threats in the use of carbapenems and colistin to fight infections.

We present Adenita, a novel software tool for the design of DNA nanostructures in a user-friendly integrated environment for molecular modeling. Adenita is capable of handling large DNA origami structures, re-use them as building blocks of new designs and provide on demand feedback, thus overcoming effectively some of the limitations of existing tools. Additionally, it integrates all major established approaches to DNA nanostructure design (DNA origami, wireframe nanostructures and DNA tiles) and allows to combine them. We show-case Adenita by re-using a large nanorod designed with Cadnano [1] to create a new nanostructure through user interactions that employ different editors to modify the original nanorod.

Artificial enzymes hold a great potential in the field of biotechnology. The currently available design strategies are limited to recruit and organise secondary and tertiary structural elements, and functional motifs of native enzymes. Here, we demonstrate an approach towards the bottom-up development of an artificial enzyme based on DNA nanotechnology. Structural analyses of the FlaI ATPase were used to identify a set of amino acids essential for the catalytic reaction. The selected amino acids were used to design four peptide-oligonucleotide conjugates (POCs). The POCs were integrated into a DNA nanostructure and reconstituted the catalytic site of FlaI. DNA origami technology was employed to maintain the relative orientations of the amino acids and their positions analogous to those in the protein catalytic site. The substrate turnover rates were found to be comparable in our artificial nanozyme (kcat = 0.3622 s-1) and the native enzyme (kcat ≈ 0.016 s-1). The nanozymes could be recycled using an ultrafiltration protocol and reused in multiple experiments allowing a reliable reproduction of the measurements. The emulation of enzymatic activity, by this novel and computable technological framework, can be of high relevance in many different areas.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and thus lower amplification efficiencies. We have developed a software workflow with three underlying algorithms that differ in their use case and specificity, allowing the complete in silico evaluation of such assays on user-derived data sets. We experimentally evaluated the method for the prediction of oligonucleotide hybridization events including resulting products and probes, self-dimers, cross-dimers and hairpins at different experimental conditions. The developed method allows explaining the observed artefacts through in silico WGS data and thermodynamic predictions. PRIMEval is available publicly at https://primeval.ait.ac.at.

Multidrug-resistant Acinetobacter baumannii (MDR-AB) is an important threat for critically ill patients. It can infect the respiratory tract, blood, soft tissues, urinary tract and central nervous system. Recently, carbapenem-resistance was observed in A. baumannii clinical isolates from Bosnia and Herzegovina. This prompted us to analyze these isolates with regards to genotypic diversity, antibiotic susceptibility and occurrence of acquired carbapenem resistance genes. Twelve carbapenem-resistant isolates were collected at a University hospital during two different periods of 2011 and 2015–2016: four isolates in 2011 and eight isolates 2015–2016 and compared to determine the dynamic changes in carbapenemase resistance mechanisms and population structure. All twelve isolates were positive for intrinsic blaOXA-51-like, nine for blaOXA-40-like and one for the blaOXA-23-like gene. ISAba1 was found upstream of blaOXA-51 in all and upstream of blaOXA-23-like gene in one isolate. Sequencing of the selected PCR products revealed the presence of OXA-72 β-lactamase (strain 1) and OXA-23 β-lactamase (strain 41). WGS of the selected isolate (AB 5) revealed the presence of blaOXA-72, chromosomal genes blaOXA-69 and blaADC. Moreover, the aac (3)-1a and aadA1 genes encoding aminoglycoside resistance, and sul1encoding sulphonamide resistance were identified. PFGE and rep-PCR revealed two clones containing highly similar isolates positive for OXA-40-like; one from 2011 and the other from 2015 to 2016. Implementation of hospital hygiene measures, screening of the patients on admission for carriage of MDR-AB, and the early and accurate detection, with restriction of antibiotic use should be recommended to control the spread of these important hospital pathogenes. To our knowledge, this is the first report of A. baumannii isolates producing carbapenem-hydrolyzing oxacillinases (CHDL) from Bosnia and Herzegovina.

Ovaj rad prikazuje upotrebu centrifugalnih kompresora i njihovu primjenu u naftno- rudarskom području te probleme i prepreke koje se javljaju tokom njihovog korištenja. Nakon uvodnog poglavlja, drugo poglavlje donosi općenitosti o kompresorima i kompresijske procese, u trećem se nalaze prednosti i mane zračnih kompresora. Zatim slijedi razlika između međuležajnog i dizajna s integriranim pogonskim zupčanikom te karakteristike kompresora. Unutar karakteristika kompresora navedene su usporedbe koeficijenta tlaka te koeficijenta protoka, karakteristična krivulja kakva jest i kakva bi trebala biti, procesi pumpanja kompresora i zaostajanja, valni ciklusi i granica vala. Nakon toga, govori se generalno o primjeni kompresora u proizvodnji nafte i plina, potom slijedi sabiranje plina, a posljednji dio vezan je uzodabir samog kompresora.

International audience; We present a novel visualization concept for DNA origami structures that integrates a multitude of representations into a Dimension and Scale Unifying Map (DimSUM). This novel abstraction map provides means to analyze, smoothly transition between, and interact with many visual representations of the DNA origami structures in an effective way that was not possible before. DNA origami structures are nanoscale objects, which are challenging to model in silico. In our holistic approach we seamlessly combine three-dimensional realistic shape models, two-dimensional diagrammatic representations, and ordered alignments in one-dimensional arrangements, with semantic transitions across many scales. To navigate through this large, two-dimensional abstraction map we highlight locations that users frequently visit for certain tasks and datasets. Particularly interesting viewpoints can be explicitly saved to optimize the workflow. We have developed DimSUM together with domain scientists specialized in DNA nanotechnology. In the paper we discuss our design decisions for both the visualization and the interaction techniques. We demonstrate two practical use cases in which our approach increases the specialists' understanding and improves their effectiveness in the analysis. Finally, we discuss the implications of our concept for the use of controlled abstraction in visualization in general.

International audience; We present an approach to represent DNA nanostructures in varying forms of semantic abstraction, describe ways to smoothly transition between them, and thus create a continuous multiscale visualization and interaction space for applications in DNA nanotechnology. This new way of observing, interacting with, and creating DNA nanostructures enables domain experts to approach their work in any of the semantic abstraction levels, supporting both low-level manipulations and high-level visualization and modifications. Our approach allows them to deal with the increasingly complex DNA objects that they are designing, to improve their features, and to add novel functions in a way that no existing single-scale approach offers today. For this purpose we collaborated with DNA nanotechnology experts to design a set of ten semantic scales. These scales take the DNA's chemical and structural behavior into account and depict it from atoms to the targeted architecture with increasing levels of abstraction. To create coherence between the discrete scales, we seamlessly transition between them in a well-defined manner. We use special encodings to allow experts to estimate the nanoscale object's stability. We also add scale-adaptive interactions that facilitate the intuitive modification of complex structures at multiple scales. We demonstrate the applicability of our approach on an experimental use case. Moreover, feedback from our collaborating domain experts confirmed an increased time efficiency and certainty for analysis and modification tasks on complex DNA structures. Our method thus offers exciting new opportunities with promising applications in medicine and biotechnology.

PURPOSE: A dramatic increase in OXA-48 β-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. ----- METHODS: Carbapenemase and other β-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. ----- RESULTS: Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 β-lactamases. In addition to the β-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. ----- CONCLUSIONS: The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, β-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

Introduction: Lyme disease is caused by the spirochete Borrelia Brugdorferi. It manifests as erythema migrans but can also cause central neuropathy, arthritis and carditis.1 Cardiac manifestations include conduction abnormalities with varying degrees of atrioventricular block and other rhythm disturbances. 2 The incidence of Lyme disease increases from west to east across Europe, with the highest incidence in Slovenia (155/100 000).3 Case report: We observed a 32-year-old male patient who presented to the Emergency Department with recurrent syncope. Electrocardiography recorded intermittent total atrioventricular block, with an average heart rate of 25/min. A temporary pacemaker (Oscor Pace 101H, Single Chamber External Pacemaker) was implanted emergently using right jugular access. He had been on Sovsko lake near Čaglin village in Požega-Slavonia county 7 days prior to admittance, after which he had fever and chills for 2 days followed by diarrhea for 1 day. We started empirical treatment with intravenous ceftriaxone due to suspected Lyme disease. No tick bites on skin were found. Results: Results of serological testing showed that IgM Lyme titer (ELISA) was positive >5.23, also CLIA IgM were positive (IgM >190, IgG >240), followed by positive IgM and IgG Western Blot. Considering these results, we continued ceftriaxone treatment according to guidelines for 6 weeks. Echocardiography demonstrated normal left and right ventricular systolic function with no valvular dysfunction. First 3 days continuous pacing was required, afterwards for 7 days only intermittent pacing in night hours was required. In the end, the patient maintained normal sinus rhythm at 65-75 bpm. Two days after, the external pacemaker and active fixation lead was removed. Conclusion: Lyme borreliosis infection can cause conduction abnormalities, including total atrioventricular block in structurally healthy heart. Conduction abnormalities are reversible with the use of proper antibiotic treatment.

The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse β-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the β-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10 4 DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of β-lactamase genes in dilutions of 10 7 cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.

U radu se analizira važnost realizacije projekta robinzonskog kampa Insula Tilagus. Dokazano je da kamp predstavlja dodatni sadržaj koji će oplemeniti turističku ponudu Dugog otoka, ukoliko se projekt realizira sukladno metodologiji projektnog menadžmenta.