Comparison of CTX-M encoding plasmids present during the early phase of the ESBL pandemic in western Sweden.

Plasmids encoding blaCTX-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 blaCTX-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 blaCTX-M-15 (n = 10), blaCTX-M-3 (n = 2) or blaCTX-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 blaCTX-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.

Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging.

DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.

A Parallelized Nanofluidic Device for High-Throughput Optical DNA Mapping of Bacterial Plasmids.

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 Escherichiacoli and Klebsiellapneumoniae, 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.

Mechanical characterization of base analogue modified nucleic acids by force spectroscopy.

We use mechanical unfolding of single DNA hairpins with modified bases to accurately assess intra- and intermolecular forces in nucleic acids. As expected, the modification stabilizes the hybridized hairpin, but we also observe intriguing stacking interactions in the unfolded hairpin. Our study highlights the benefit of using base-modified nucleic acids in force-spectroscopy.

Shining light on single-strand lesions caused by the chemotherapy drug bleomycin.

Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.

Identity of blaCTX-M Carrying Plasmids in Sequential ESBL-E. coli Isolates from Patients with Recurrent Urinary Tract Infections.

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.

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging.

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.

Optical maps of plasmids as a proxy for clonal spread of MDR bacteria: a case study of an outbreak in a rural Ethiopian hospital.

OBJECTIVES: MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements. We believe that this method has the potential to be a tool of pivotal importance for MDR control. METHODS: Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria. RESULTS: ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles. CONCLUSIONS: ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low- and middle-income countries.

Molecular Epidemiology of OXA-48 and NDM-1 Producing Enterobacterales Species at a University Hospital in Tehran, Iran, Between 2015 and 2016.

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 bla NDM-1 and bla OXA-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 bla NDM-1 and bla OXA-48 were successfully transferred to an E. coli K12-recipient strain. The bla OXA-48 gene was located on an IncL/M conjugative plasmid, while the bla NDM-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 bla NDM-1 and bla OXA-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.

Annealing of ssDNA and compaction of dsDNA by the HIV-1 nucleocapsid and Gag proteins visualized using nanofluidic channels.

The nucleocapsid protein NC is a crucial component in the human immunodeficiency virus type 1 life cycle. It functions both in its processed mature form and as part of the polyprotein Gag that plays a key role in the formation of new viruses. NC can protect nucleic acids (NAs) from degradation by compacting them to a dense coil. Moreover, through its NA chaperone activity, NC can also promote the most stable conformation of NAs. Here, we explore the balance between these activities for NC and Gag by confining DNA-protein complexes in nanochannels. The chaperone activity is visualized as concatemerization and circularization of long DNA via annealing of short single-stranded DNA overhangs. The first ten amino acids of NC are important for the chaperone activity that is almost completely absent for Gag. Gag condenses DNA more efficiently than mature NC, suggesting that additional residues of Gag are involved. Importantly, this is the first single DNA molecule study of full-length Gag and we reveal important differences to the truncated Δ-p6 Gag that has been used before. In addition, the study also highlights how nanochannels can be used to study reactions on ends of long single DNA molecules, which is not trivial with competing single DNA molecule techniques.

Nanofluidic Fluorescence Microscopy (NFM) for real-time monitoring of protein binding kinetics and affinity studies.

Kinetic monitoring of protein interactions offers insights to their corresponding functions in cellular processes. Surface plasmon resonance (SPR) is the current standard tool used for label-free kinetic assays; however, costly and sophisticated setups are required, decreasing its accessibility to research laboratories. We present a cost-effective nanofluidic-based immunosensor for low-noise real-time kinetic measurement of fluorescent-labeled protein binding. With the combination of fluorescence microscopy and reversed buffer flow operation, association and dissociation kinetics can be accessed in one single experiment without extra buffer loading step, which results in a simplified operation and reduced time of analysis compared to typical microfluidic immunoassays. Kinetic constants of two representative protein-ligand binding pairs (streptavidin/biotin; IgG/anti-IgG) were quantified. The good agreement of extracted rate constants with literature values and analogous SPR measurements indicates that this approach is applicable to study protein interactions of medium- and high-affinities with a limit of detection down to 1 pM, regardless of the analyte size.

Multiplexed immunosensing and kinetics monitoring in nanofluidic devices with highly enhanced target capture efficiency.

Nanofluidic devices promise high reaction efficiency and fast kinetic responses due to the spatial constriction of transported biomolecules with confined molecular diffusion. However, parallel detection of multiple biomolecules, particularly proteins, in highly confined space remains challenging. This study integrates extended nanofluidics with embedded protein microarray to achieve multiplexed real-time biosensing and kinetics monitoring. Implementation of embedded standard-sized antibody microarray is attained by epoxy-silane surface modification and a room-temperature low-aspect-ratio bonding technique. An effective sample transport is achieved by electrokinetic pumping via electroosmotic flow. Through the nanoslit-based spatial confinement, the antigen-antibody binding reaction is enhanced with ∼100% efficiency and may be directly observed with fluorescence microscopy without the requirement of intermediate washing steps. The image-based data provide numerous spatially distributed reaction kinetic curves and are collectively modeled using a simple one-dimensional convection-reaction model. This study represents an integrated nanofluidic solution for real-time multiplexed immunosensing and kinetics monitoring, starting from device fabrication, protein immobilization, device bonding, sample transport, to data analysis at Péclet number less than 1.

Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices.

Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein-DNA interaction sites.

Biologically active components and nutraceuticals in the Monascus-fermented rice: a review.

Monascus-fermented rice has traditionally been used as a natural food colorant and food preservative of meat and fish for centuries. It has recently become a popular dietary supplement because of many of its bioactive constituents being discovered, including a series of active drug compounds, monacolins, indicated as the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors for reducing serum cholesterol level. The controversy of its safety has been provoked because a mycotoxin, citrinin, is also produced along with the Monascus secondary metabolites by certain strains or under certain cultivation conditions. This review introduces the basic production process and addresses on the compounds with bioactive functions. Current advances in avoiding the harmful ingredient citrinin are also discussed.

Ankaflavin from Monascus-fermented red rice exhibits selective cytotoxic effect and induces cell death on Hep G2 cells.

Monascus-fermented red rice has traditionally been used as a natural food colorant or food preservative of meat and fish for centuries. Recently, it has become a popular dietary supplement due to many of its bioactive constituents being discovered. Commercial Monascus-fermented red rice was used in this study. According to the cell-based cytotoxicity assay, a compound with selective cytotoxicity was found and identified as ankaflavin. Ankaflavin was found to be toxic to human cancer cell lines Hep G2 and A549 with a similar IC50 value of 15 microg/mL, while it posed no significant toxicity to normal MRC-5 and WI-38 cells at the same concentration. For elucidating the possible mode of cell death, Hep G2 cells were treated with ankaflavin for 48 h to examine the morphological change of the chromatin. Chromosomal condensation and fragmentation were found, and a significant sub-G1 peak was found by flow cytometry. Apoptosis was therefore suggested as the possible mechanism. Monascin, an analogue of ankaflavin, was also tested in this study. However, it showed no cytotoxicity and did not induce death of Hep G2 cells.