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Abstract Plasmids encoding bla CTX-M genes have greatly shaped the evolution of E. coli producing extended-spectrum beta-lactamases (ESBL-E. coli) and adds to the global threat of multiresistant bacteria by promoting horizontal gene transfer (HGT). Here we screened the similarity of 47 bla CTX-M -encoding plasmids, from 45 epidemiologically unrelated and disperse ESBL-E. coli strains, isolated during the early phase (2009–2014) of the ESBL pandemic in western Sweden. Using optical DNA mapping (ODM), both similar and rare plasmids were identified. As many as 57% of the plasmids formed five ODM-plasmid groups of at least three similar plasmids per group. The most prevalent type (28%, IncIl, pMLST37) encoded bla CTX-M-15 (n = 10), bla CTX-M-3 (n = 2) or bla CTX-M-55 (n = 1). It was found in isolates of various sequence types (STs), including ST131. This could indicate ongoing local HGT as whole-genome sequencing only revealed similarities with a rarely reported, IncIl plasmid. The second most prevalent type (IncFII/FIA/FIB, F1:A2:B20) harboring bla CTX-M-27, was detected in ST131-C1-M27 isolates, and was similar to plasmids previously reported for this subclade. The results also highlight the need for local surveillance of plasmids and the importance of temporospatial epidemiological links so that detection of a prevalent plasmid is not overestimated as a potential plasmid transmission event in outbreak investigations.

We introduce the concept photophysical image analysis (PIA) and an associated pipeline for unsupervised probabilistic image thresholding for images recorded by electron-multiplying charge-coupled device (EMCCD) cameras. We base our approach on a closed-form analytic expression for the characteristic function (Fourier-transform of the probability mass function) for the image counts recorded in an EMCCD camera, which takes into account both stochasticity in the arrival of photons at the imaging camera and subsequent noise induced by the detection system of the camera. The only assumption in our method is that the background photon arrival to the imaging system is described by a stationary Poisson process (we make no assumption about the photon statistics for the signal). We estimate the background photon statistics parameter, λbg, from an image which contains both background and signal pixels by use of a novel truncated fit procedure with an automatically determined image count threshold. Prior to this, the camera noise model parameters are estimated using a calibration step. Utilizing the estimates for the camera parameters and λbg, we then introduce a probabilistic thresholding method, where, for the first time, the fraction of misclassified pixels can be determined a priori for a general image in an unsupervised way. We use synthetic images to validate our a priori estimates and to benchmark against the Otsu method, which is a popular unsupervised non-probabilistic image thresholding method (no a priori estimates for the error rates are provided). For completeness, we lastly present a simple heuristic general-purpose segmentation method based on the thresholding results, which we apply to segmentation of synthetic images and experimental images of fluorescent beads and lung cell nuclei. Our publicly available software opens up for fully automated, unsupervised, probabilistic photophysical image analysis.

Nyblom, Johnning et al. develop an optical DNA mapping approach for bacterial strain typing of patient samples. They demonstrate rapid identification of clinically relevant E. coli and K. pneumoniae strains, without the need for cultivation.

Using fibroblast growth factor 2 (FGF-2) induced sense and antisense transcripts IER3 and IER3-AS1 as a model system, authors highlight the role of HnRNPK in regulating double strand RNA formation in the loci with overlapping transcripts.

Abstract Antimicrobial resistance (AMR) is a fast-growing threat to global health. The genes conferring AMR to bacteria are often located on plasmids, circular extrachromosomal DNA molecules that can be transferred between bacterial strains and species. Therefore, effective methods to characterize bacterial plasmids and detect the presence of resistance genes can assist in managing AMR, for example, during outbreaks in hospitals. However, existing methods for plasmid analysis either provide limited information or are expensive and challenging to implement in low-resource settings. Herein, we present a simple assay based on CRISPR/Cas9 excision and DNA combing to detect antimicrobial resistance genes on bacterial plasmids. Cas9 recognizes the gene of interest and makes a double-stranded DNA cut, causing the circular plasmid to linearize. The change in plasmid configuration from circular to linear, and hence the presence of the AMR gene, is detected by stretching the plasmids on a glass surface and visualizing by fluorescence microscopy. This single-molecule imaging based assay is inexpensive, fast, and in addition to detecting the presence of AMR genes, it provides detailed information on the number and size of plasmids in the sample. We demonstrate the detection of several β-lactamase-encoding genes on plasmids isolated from clinical samples. Furthermore, we demonstrate that the assay can be performed using standard microbiology and clinical laboratory equipment, making it suitable for low-resource settings.

ABSTRACT: Objectives: The burden of antimicrobial resistance and spread of epidemic clones are rarely reported from low-income countries. We aimed to investigate the genome-based epidemiology of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) at a tertiary hospital in Jimma, Ethiopia. Methods: Bacteria were isolated from clinical specimens at Jimma Medical Center and subjected to species identification (MALDI-TOF), antimicrobial susceptibility testing (disk diffusion) and whole-genome sequencing (Illumina, HiSeq2500). Genomic data analysis was performed using EnteroBase and Center for Genomic Epidemiology bioinformatics pipelines. A maximum likelihood tree was generated using FastTree/2.1.8 based on single nucleotide polymorphisms (SNPs) in shared genomic regions to identify transmission clusters. Results: Escherichia coli isolates (n = 261) were collected from 1087 single non-duplicate clinical specimens over a 5-month period in 2016. The prevalence of ESBL-EC was 54.8% (143/261), 96% of which were resistant to multiple antibiotic classes. The blaCTX-M-15 ESBL gene was present in 88.4.% of isolates (122/138). Genes conferring resistance to aminoglycosides and ciprofloxacin [aac(6′)-Ib-cr, 62.3% (86/138)], phenicols [catB3, 56.5% (78/138)], sulfonamides [sul1, 68.1% (94/138), trimethoprim [dfrA17, 58.0% (80/138)] and macrolides [mph(A), 67.4% (93/138) were detected. The most prevalent sequence types were ST410 (23%), ST648 (17%), ST131 (10%) and ST167 (7%). Isolates of the same sequence type collected from different units of the hospital were highly similar in the SNP analysis. Conclusion: A high prevalence of ESBLs and dissemination of blaCTX-M-15 through multiple high-risk E. coli clones was detected. Nosocomial spread of multidrug-resistant ESBL-EC within the hospital puts vulnerable patients at risk of difficult-to-treat infections.

Abstract Optical nanoantennas provide control of light at the nanoscale, which makes them important for diverse areas ranging from photocatalysis and flat metaoptics to sensors and biomolecular tweezing. They have traditionally been limited to metallic and dielectric nanostructures that sustain plasmonic and Mie resonances, respectively. More recently, nanostructures of organic J‐aggregate excitonic materials have been proposed capable of also supporting nanooptical resonances, although their advance has been hampered from difficulty in nanostructuring. Here, the authors present the realization of organic J‐aggregate excitonic nanostructures, using nanocylinder arrays as model system. Extinction spectra show that they can sustain both plasmon‐like resonances and dielectric resonances, owing to the material providing negative and large positive permittivity regions at the different sides of its exciton resonance. Furthermore, it is found that the material is highly anisotropic, leading to hyperbolic and elliptic permittivity regions. Nearfield analysis using optical simulation reveals that the nanostructures therefore support hyperbolic localized surface exciton resonances and elliptic Mie resonances, neither of which has been previously demonstrated for this type of material. The anisotropic nanostructures form a new type of optical nanoantennas, which combined with the presented fabrication process opens up for applications such as fully organic excitonic metasurfaces.

ObjectivesCarbapenemase-producing Enterobacterales (CPE) are high priority targets of global antimicrobial surveillance. Herein, we determined the colonization rate of CPE on admission to intensive care units in Vientiane, Lao PDR in August-September 2019.MethodsData regarding clinical conditions, infection control, and antibiotic usage were collected during admission. Rectal swab samples (n = 137) collected during admission were inoculated to selective chromogenic agars, followed by confirmatory tests for extended-spectrum beta-lactamases and carbapenemases. All CPE isolates were sequenced on Illumina (HiSeq2500), reads assembled using SPAdes 3.13, and the draft genomes used to query a database (https://www.genomicepidemiology.org) for resistome, plasmid replicons, and sequence types (ST). Optical DNA mapping (ODM) was used to characterize plasmids and to determine location of resistance genes. Minimum spanning tree was generated using the Bacterial Isolate Genome Sequence database (BIGSdb) and annotated using iTOL.ResultFrom 47 Enterobacterales isolated on selective agars, K. pneumoniae (25/47) and E. coli (12/47) were the most prevalent species, followed by K aerogenes (2/47), K. variicola (1/47), and K. oxytoca (1/47). The overall prevalence of ESBLs was 51.0%; E. coli 83.3% (10/12) and Klebsiella spp. 41.3% (12/29). Twenty percent of the K. pneumoniae (5/25) isolates were carbapenem-resistant, and 4/5 contained the blaNDM-1 gene. All blaNDM-1 isolates belonged to ST147 and were indistinguishable with cgMLST. ODM showed that the blaNDM-1 gene was located on identical plasmids in all isolates.ConclusionThe prevalence of ESBL-producing Enterobacterales was high, while carbapenemases were less common. However, the detection of clonal dissemination of blaNDM-1-producing K. pneumoniae isolates in one of the intensive care units calls for vigilance. Stringent infection prevention and antimicrobial stewardship strategies are highly important measures.

Single nanoparticle catalysis studies enable structure–function correlations beyond averaged properties. Here, the authors present a versatile nanofluidics- and fluorescence-based platform for highly parallelized single particle catalysis in solution.

Carbapenem-resistant Klebsiella pneumoniae are a major global threat in healthcare facilities. The propagation of carbapenem resistance determinants can occur through vertical transmission, with genetic elements being transmitted by the host bacterium, or by horizontal transmission, with the same genetic elements being transferred among distinct bacterial hosts. This work aimed to track carbapenem resistance transmission by K. pneumoniae in a healthcare facility. The study involved a polyphasic approach based on conjugation assays, resistance phenotype and genotype analyses, whole genome sequencing, and plasmid characterization by pulsed field gel electrophoresis and optical DNA mapping. Out of 40 K. pneumoniae clinical isolates recovered over two years, five were carbapenem- and multidrug-resistant and belonged to multilocus sequence type ST147. These isolates harboured the carbapenemase encoding blaKPC-3 gene, integrated in conjugative plasmids of 140 kbp or 55 kbp, belonging to replicon types incFIA/incFIIK or incN/incFIIK, respectively. The two distinct plasmids encoding the blaKPC-3 gene were associated with distinct genetic lineages, as confirmed by optical DNA mapping and whole genome sequence analyses. These results suggested vertical (bacterial strain-based) transmission of the carbapenem-resistance genetic elements. Determination of the mode of transmission of antibiotic resistance in healthcare facilities, only possible based on polyphasic approaches as described here, is essential to control resistance propagation.

Large-scale genomic alterations play an important role in disease, gene expression, and chromosome evolution. Optical DNA mapping (ODM), commonly categorized into sparsely-labelled ODM and densely-labelled ODM, provides sequence-specific continuous intensity profiles (DNA barcodes) along single DNA molecules and is a technique well-suited for detecting such alterations. For sparsely-labelled barcodes, the possibility to detect large genomic alterations has been investigated extensively, while densely-labelled barcodes have not received as much attention. In this work, we introduce HMMSV, a hidden Markov model (HMM) based algorithm for detecting structural variations (SVs) directly in densely-labelled barcodes without access to sequence information. We evaluate our approach using simulated data-sets with 5 different types of SVs, and combinations thereof, and demonstrate that the method reaches a true positive rate greater than 80% for randomly generated barcodes with single variations of size 25 kilobases (kb). Increasing the length of the SV further leads to larger true positive rates. For a real data-set with experimental barcodes on bacterial plasmids, we successfully detect matching barcode pairs and SVs without any particular assumption of the types of SVs present. Instead, our method effectively goes through all possible combinations of SVs. Since ODM works on length scales typically not reachable with other techniques, our methodology is a promising tool for identifying arbitrary combinations of genomic alterations.

Optical DNA mapping (ODM) is based on fluorescent labeling, stretching and imaging of single DNA molecules to obtain sequence-specific fluorescence profiles, DNA barcodes. These barcodes can be mapped to theoretical counterparts obtained from DNA reference sequences, which in turn allow for DNA identification in complex samples and for detecting structural changes in individual DNA molecules. There are several types of DNA labeling schemes for ODM and for each labeling type one or several types of match scoring methods are used. By combining the information from multiple labeling schemes one can potentially improve mapping confidence; however, combining match scores from different labeling assays has not been implemented yet. In this study, we introduce two theoretical methods for dealing with analysis of DNA molecules with multiple label types. In our first method, we convert the alignment scores, given as output from the different assays, into p-values using carefully crafted null models. We then combine the p-values for different label types using standard methods to obtain a combined match score and an associated combined p-value. In the second method, we use a block bootstrap approach to check for the uniqueness of a match to a database for all barcodes matching with a combined p-value below a predefined threshold. For obtaining experimental dual-labeled DNA barcodes, we introduce a novel assay where we cut plasmid DNA molecules from bacteria with restriction enzymes and the cut sites serve as sequence-specific markers, which together with barcodes obtained using the established competitive binding labeling method, form a dual-labeled barcode. All experimental data in this study originates from this assay, but we point out that our theoretical framework can be used to combine data from all kinds of available optical DNA mapping assays. We test our multiple labeling frameworks on barcodes from two different plasmids and synthetically generated barcodes (combined competitive-binding- and nick-labeling). It is demonstrated that by simultaneously using the information from all label types, we can substantially increase the significance when we match experimental barcodes to a database consisting of theoretical barcodes for all sequenced plasmids.

Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents.

Carbapenem-resistant Enterobacterales (CRE) is an increasing problem worldwide. Here, we examined the clonal relatedness of 71 non-repetitive CRE isolates collected in a university hospital in Tehran, Iran, between February 2015 and March 2016. Pulsed-field gel electrophoresis (PFGE) and MLST were used for epidemiological analysis. Screening for antibiotic resistance genes, PCR-based replicon typing, conjugation experiments, and optical DNA mapping were also performed. Among all 71 isolates, 47 isolates of Klebsiella pneumoniae (66.2%), eight Escherichia coli (11.2%), five Serratia marcescens (7%), and two Enterobacter cloacae (2.8%) harbored blaNDM–1 and blaOXA–48 genes together or alone. PFGE analysis revealed that most of the OXA-48- and NDM-1-producing K. pneumoniae and all of OXA-48-producing S. marcescens were clonally related, while all eight E. coli and two E. cloacae isolates were clonally unrelated. The predominant clones of carbapenemase-producing K. pneumoniae associated with outbreaks within the hospital were ST147 (n = 13) and ST893 (n = 10). Plasmids carrying blaNDM–1 and blaOXA–48 were successfully transferred to an E. coli K12-recipient strain. The blaOXA–48 gene was located on an IncL/M conjugative plasmid, while the blaNDM–1 gene was located on both IncFII ∼86-kb to ∼140-kb and IncA/C conjugative plasmids. Our findings provide novel epidemiologic data on carbapenemase-producing Enterobacterales (CPE) in Iran and highlight the importance of horizontal gene transfer in the dissemination of blaNDM–1 and blaOXA–48 genes. The occurrence and transmission of distinct K. pneumoniae clones call for improved infection control to prevent further spread of these pathogens in Iran.

Ananda Devi and J.M.G. Le Clézio have both close connections to France as well as to Mauritius. While the former is rather qualified as a Mauritian author, the latter is rather seen as « French ». In this paper, I explore some mechanisms behind the attribution of a nationality to an author, by using a modified version of Jorge Caldéron’s critical questions concerning the « Pour une littérature-monde en français » manifesto. I find that there is no criterion for defining the national identity of an author, that couldn’t be questioned by finding counter-examples. Ananda Devi et J.M.G. Le Clézio ont tous deux des liens forts aussi bien avec la France qu’avec l’île Maurice. L’une est pourtant plutôt qualifiée de « mauricienne », tandis que l’autre est plutôt qualifié de « français ». À l’aide d’une version modifiée des questions posées par Jorge Caldéron dans sa critique du manifeste « Pour une littérature-monde en français », nous explorons les enjeux de l’attribution à ces deux auteurs d’une nationalité perçue plutôt qu’une autre. Nous trouvons que, quoi que l’on pose comme critère, il y a toujours des contre-exemples qui la remettent en question.

Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids—circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

Pre-miRNA-377 is a hairpin-shaped regulatory RNA associated with heart failure. Here, we use single-molecule optical tweezers to unzip pre-miRNA-377 and study its stability and dynamics. We show that magnesium ions have a strong stabilizing effect, and that sodium ions stabilize the hairpin more than potassium ions. The hairpin unfolds in a single step, regardless of buffer composition. Interestingly, hairpin folding occurs either in a single step (type 1) or through the formation of intermediates, in multiple steps (type 2) or gradually (type 3). Type 3 occurs only in the presence of both sodium and magnesium, while type 1 and 2 take place in all buffers, with type 1 being the most prevalent. By reducing the size of the native hairpin loop from fourteen to four nucleotides, we demonstrate that the folding heterogeneity originates from the large size of the hairpin loop. Further, while efficient pre-miRNA-377 binders are lacking, we demonstrate that the recently developed C2 ligand displays bimodal activity: it enhances the mechanical stability of the pre-miRNA-377 hairpin and perturbs its folding. The knowledge regarding pre-miRNA stability and dynamics that we provide is important in understanding its regulatory function and how it can be modulated to achieve a therapeutic effect, e.g., in heart failure treatment.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) in patients admitted to hospitals pose a great challenge to treatment. The genes causing resistance to carbapenems are mostly found in plasmids, mobile genetic elements that can spread easily to other bacterial strains, thus exacerbating the problem. Here, we studied 27 CR-KP isolates collected from different types of samples from 16 patients admitted to the medical ward at Siriraj Hospital in Bangkok, Thailand, using next generation sequencing (NGS) and optical DNA mapping (ODM). The majority of the isolates belonged to sequence type (ST) 16 and are described in detail herein. Using ODM, we identified the plasmid carrying the blaNDM-1 gene in the ST16 isolates and the plasmids were very similar, highlighting the possibility of using ODM of plasmids as a surrogate marker of nosocomial spread of bacteria. We also demonstrated that ODM could identify that the blaCTX-M-15 and blaOXA-232 genes in the ST16 isolates were encoded on separate plasmids from the blaNDM-1 gene and from each other. The other three isolates belonged to ST147 and each of them had distinct plasmids encoding blaNDM-1.

Plasmid-mediated multidrug resistance in E. coli is becoming increasingly prevalent. Considering this global threat to human health, it is important to understand how plasmid-mediated resistance spreads. From a cohort of 123 patients with recurrent urinary tract infections (RUTI) due to extended spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL E. coli), only five events with a change of ESBL E. coli strain between RUTI episodes were identified. Their blaCTX-M encoding plasmids were compared within each pair of isolates using optical DNA mapping (ODM) and PCR-based replicon typing. Despite similar blaCTX-M genes and replicon types, ODM detected only one case with identical plasmids in the sequential ESBL E. coli strains, indicating that plasmid transfer could have occurred. For comparison, plasmids from seven patients with the same ESBL E. coli strain reoccurring in both episodes were analyzed. These plasmids (encoding blaCTX-M-3, blaCTX-M-14, and blaCTX-M-15) were unaltered for up to six months between recurrent infections. Thus, transmission of blaCTX-M plasmids appears to be a rare event during the course of RUTI. Despite the limited number (n = 23) of plasmids investigated, similar blaCTX-M-15 plasmids in unrelated isolates from different patients were detected, suggesting that some successful plasmids could be associated with specific strains, or are more easily transmitted.

Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence–based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches. In addition, designing lab-on-a-chip systems based on OM is now feasible and will allow the integrated and automated microbiological analysis of biological fluids. Here, we review the basic technology of OM, detail the current state of the art of the field, and look ahead to possible future developments in OM technology for microbiological applications.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ABSTRACT The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings. IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.

There are contradictory reports in the literature regarding the anti-bacterial activity of graphene, graphene oxide (GO) and reduced graphene oxide (rGO). This controversy is mostly due to variations in key parameters of the reported experiments, like: type of substrate, form of graphene, number of layers, type of solvent and most importantly, type of bacteria. Here, we present experimental data related to bacterial response to GO and rGO integrated in solid agar-based nutrient plates—a standard set-up for bacterial growth that is widely used by microbiologists. Bacillus subtilis and Pseudomonas aeruginosa strains were used for testing bacterial growth. We observed that plate-integrated rGO showed strong anti-bacterial activity against both bacterial species. By contrast, plate-integrated GO was harmless to both bacteria. These results reinforce the notion that the response of bacteria depends critically on the type of graphene material used and can vary dramatically from one bacterial strain to another, depending on bacterial physiology.

The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard "foreign" contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.

Extracellular polymeric substances (EPS) produced by bacteria form a matrix supporting the complex three-dimensional architecture of biofilms. This EPS matrix is primarily composed of polysaccharides, proteins and extracellular DNA. In addition to supporting the community structure, the EPS matrix protects bacterial biofilms from the environment. Specifically, it shields the bacterial cells inside the biofilm, by preventing antimicrobial agents from getting in contact with them, thereby reducing their killing effect. New strategies for disrupting the formation of the EPS matrix can therefore lead to a more efficient use of existing antimicrobials. Here we examined the mechanism of the known effect of vitamin C (sodium ascorbate) on enhancing the activity of various antibacterial agents. Our quantitative proteomics analysis shows that non-lethal concentrations of vitamin C inhibit bacterial quorum sensing and other regulatory mechanisms underpinning biofilm development. As a result, the EPS biosynthesis in reduced, and especially the polysaccharide component of the matrix is depleted. Once the EPS content is reduced beyond a critical point, bacterial cells get fully exposed to the medium. At this stage, the cells are more susceptible to killing, either by vitamin C-induced oxidative stress as reported here, or by other antimicrobials or treatments.

In optical DNA mapping technologies sequence-specific intensity variations (DNA barcodes) along stretched and stained DNA molecules are produced. These "fingerprints" of the underlying DNA sequence have a resolution of the order one kilobasepairs and the stretching of the DNA molecules are performed by surface adsorption or nano-channel setups. A post-processing challenge for nano-channel based methods, due to local and global random movement of the DNA molecule during imaging, is how to align different time frames in order to produce reproducible time-averaged DNA barcodes. The current solutions to this challenge are computationally rather slow. With high-throughput applications in mind, we here introduce a parameter-free method for filtering a single time frame noisy barcode (snap-shot optical map), measured in a fraction of a second. By using only a single time frame barcode we circumvent the need for post-processing alignment. We demonstrate that our method is successful at providing filtered barcodes which are less noisy and more similar to time averaged barcodes. The method is based on the application of a low-pass filter on a single noisy barcode using the width of the Point Spread Function of the system as a unique, and known, filtering parameter. We find that after applying our method, the Pearson correlation coefficient (a real number in the range from -1 to 1) between the single time-frame barcode and the time average of the aligned kymograph increases significantly, roughly by 0.2 on average. By comparing to a database of more than 3000 theoretical plasmid barcodes we show that the capabilities to identify plasmids is improved by filtering single time-frame barcodes compared to the unfiltered analogues. Since snap-shot experiments and computational time using our method both are less than a second, this study opens up for high throughput optical DNA mapping with improved reproducibility.

The 2,6,10-tris(dialkylamino)trioxatriangulenium dyes (ATOTA+) are highly stabilised cationic chromophores with D3hsymmetry. The symmetry gives rise to a degeneracy of the main electronic transition. In low polarity solvents significant splitting of this degenerate transition is observed and assigned to ion pair formation. Ion pairing of the 2,6,10-tris(dioctylamino)trioxatriangulenium ion with Cl−, BF4−, PF6−and TRISPHAT anions was studied using absorption spectroscopy. A clear correlation is found between the size of the anion and the splitting of the ATOTA+transitions. In benzene the Cl−salt displays a splitting of 1955 cm−1, while the salt of the much larger TRISPHAT ion has a splitting of 1543 cm−1. TD-DFT calculations confirm the splitting of the states and provide a detailed insight into the electronic structure of the ion pairs. The different degree of splitting in different ion pairs is found to correlate with the magnitude of the electric field generated in each ion pair, thus leading to the conclusion that the effect seen is an internal Stark effect. By insertion of an amphiphilic derivative of the ATOTA+chromophore in an oriented lamellar liquid crystal, it was possible to resolve the two bands of the double peak spectrum and show their perpendicular orientation in the molecular framework, as predicted by the calculations. [ABSTRACT FROM AUTHOR]

Structural and optical properties of multilayer Langmuir−Blodgett (LB) films of two amphiphilic carbenium salts 2-didecylamino-6,10-bis(dimethylamino)-4,8,12-trioxatriangulenium hexafluorophosphate (ATOTA-1) and 2,6-bis(decylmethylamino)-10-dimethylamino-4,8,12-trioxatriangulenium hexafluorophosphate (ATOTA-2) are described. The LB films were prepared on lipophilic glass by standard vertical dipping. Grazing incidence X-ray diffraction (GIXD) measurements show that the planar organic cores, in spite of their positive charge, form closely packed columns with a repeating distance of ∼3.45 Å. Specular X-ray reflectivity (SXR) reveals the LB multilayers to consist of Y-type bilayers with thickness 31 Å for ATOTA-1 and 41 Å for ATOTA-2. This significant difference is ascribed to the different packing motifs of the alkyl chains in the two LB films. GIXD and polarized UV−vis absorption and emission spectroscopy show that the columnar aggregates in the LB films are oriented along the dipping direction. This alignment is attributed to shear effects during LB transfer. The main absorption band of the LB films is blue-shifted compared to that in solution, while the fluorescence is red-shifted by more than 100 nm. These findings suggest the presence of H-aggregates in agreement with the cofacial packing derived from the X-ray measurements. Polarized absorption spectroscopy with variable angle of incidence was used to resolve two perpendicular optical transitions in the visible range, one at 460 nm polarized perpendicular to the columnar direction, in the plane of the film, and one at 420 nm polarized along the film normal. [ABSTRACT FROM AUTHOR]

We here report on the formation of well-ordered 2D gold nanostructures at the air/water interface. Spreading a mixture of alkanethiol-capped gold nanoparticles (AuNPs) and an amphiphilic poly(p-phenylene) on a water surface and compressing the mixture to a surface pressure of 40 mN/m lead to the formation of a network of well-ordered gold nanostructures. The structures are transferred horizontally (Langmuir−Schäfer) onto a solid substrate and investigated with TEM, AFM, and X-ray reflectivity, showing a pattern that is repeating over several micrometers. AFM and X-ray reflectivity data at different surface pressures reveal that the polymer is lifting the AuNPs 1.5−2 nm in the vertical direction, away from the polymer layer, when the pressure is increased from 20 to 40 mN/m. [ABSTRACT FROM AUTHOR]

We here exploit the recently reported thermodynamic preference for poly(dAdT)2over mixed-sequence calf thymus (ct) DNA of two binuclear ruthenium complexes, --bidppz(bipy)4Ru24(B) and --bidppz(phen)4Ru24(P), that bind to DNA by threading intercalation, to determine their intrinsic dissociation rates. After adding poly(dAdT)2as a sequestering agent to Bor Pbound to ct-DNA, the observed rate of change in luminescence upon binding to the polynucleotide reflects the rate of dissociation from the mixed sequence. The activation parameters for the threading and dissociation rate constants allow us for the first time to characterize the thermodynamics of the exceedingly slow threading intercalation equilibrium of Band Pwith ct-DNA. The equilibrium is found to be endothermic by 33 and 76 kJ/mol, respectively, and the largest part of the enthalpy difference between the complexes originates from the forward threading step. At physiological temperature (37 °C) Band Phave dissociation half-lives of 18 and 38 h, respectively. This is to our knowledge the slowest dissociating noncovalently bound DNA−drug reported. SDS sequestration is the traditional method for determination of rate constants for cationic drugs dissociating from DNA. However, the rates may be severely overestimated for slowly dissociating molecules due to unwanted catalysis by the SDS monomers and micelles. Having determined the intrinsic dissociation rates with poly(dAdT)2as sequestering agent, we find that the catalytic effect of SDS on the dissociation rate may be up to a factor of 60, and that the catalysis is entropy driven. A simple kinetic model for the SDS concentration dependence of the apparent dissociation rate suggests an intermediate that involves both micelles and DNA-threaded complex. [ABSTRACT FROM AUTHOR]

The DNA binding conformation and the photophysical properties of the semiflexible binuclear ruthenium complex -bidppz(phen)4Ru24(2) were studied with optical spectroscopy and compared to the rigid, planar homologue in syn conformation -dtpf(phen)4Ru24(3) and the parent “light-switch” complex Ru(phen)2dppz2(1). Comparison of calculated and observed absorption bands of the bridging ligand, bidppz, confirm earlier suggestions that 2is significantly nonplanar, both free in solution and when intercalated into poly(dAdT)2, but the conclusion that the intercalated conformation is an anti rotamer is not substantiated by comparison of linear and circular dichroism spectra of 2and 3. The behavior of the emission quantum yield as a function of temperature is similar for the two binuclear complexes 2and 3in different protic solvents, and a quantitative analysis suggests that, in solution, the solvent is more strongly hydrogen bonded to the excited state of 2than to 1. However, the observation that for 2the radiative rate constant increases to a value similar to 1upon intercalation into DNA suggests that the difference between 1and 2in accepting hydrogen bonds is less pronounced when intercalated. [ABSTRACT FROM AUTHOR]

We demonstrate that the detected emission intensity from YOYO-labeled DNA molecules confined in 180 nm deep Si/SiO2nanofunnels changes significantly and not monotonically with the width of the funnel. This effect may be of importance for quantitative fluorescence microscopy and for experiments with a tight photon budget. [ABSTRACT FROM AUTHOR]