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

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

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

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

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

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 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

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

Biosensors in Extended Nanofluidic Device

微流道具有低樣品體積需求、反應速度快、微型化與自動化的優勢，已被廣泛應用於生物醫學檢驗。近年來技術的進展，更將流道的尺度推到奈米等級，促使生物分子間的反應距離更接近其擴散距離，達到極高(接近100%)的反應效率。本研究結合表面修飾與室溫晶片封裝方法，建立了一個結合抗體陣列的奈米流道生物感測平台。藉由螢光標記，即時觀察奈米流道內抗原與抗體的結合反應。由於奈米流道內的高反應效率特性，常見上游的抗體陣列抓取了多數的螢光抗原，造成下游生成無螢光訊號之無反應區域。我們模擬流道內的流體輸送與反應，重現奈米流道內反應的空間分佈，並開發了奈米流道內特用的一維模型，簡化並加快運算的效率。本研究可望進一步開發為微小化、同時檢測多種生物標記之檢驗平台。Nano/microfluidic devices have shown their superior capabilities in various applications. As compared to conventional biochemical assays, nano/microfluidic devices pose minimized dimensions, reduce the required sample consumption and shorten the overall assay time. Recent progress in lab-on-a-chip technologies further allows the miniaturization of fluidic channels to nanoscale level where molecular reactions become extremely efficiently as the inter-molecular distance are confined to be comparable to the molecular diffusion distance. This study represents a methodology to implement antibody microarray into extended nanoslits. Through moderate surface modification, fluidic chips are compatible with microarray spotting and the following chip encapsulation procedure. A room temperature chip-bonding process using polysilsesquioxane as gasket layer was integrated in the procedure to preserve the activities of the immobilized biomolecules. Furthermore, the antigen-antibody binding kinetics in the confined space was observed via real-time fluorescence imaging. In addition to antibody specificity preservation, extremely high reaction efficiency was observed as upstream microspots collect most fluorescently labeled target molecules and creates a dark depletion zone in the downstream. The kinetics was simulated through a finite element model and also a simplified one-dimensional convection-reaction model, which confirms that diffusion is efficient in the confined space. This work provides a combined strategy to incorporate protein microarrays within extended nanoslits, and a side-by-side simulation to study the kinetics

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

Nanofluidic optical DNA mapping for rapid identification of antibiotics resistant plasmids

Bacterial resistance to antibiotics has become a major threat to health worldwide. In 2014, the World Health Organization (WHO) classified antibiotic resistance as one of the major threats to human health.1 As the resistance genes are frequently transmitted via mobile genetic elements, such as plasmids, a method to rapidly detect and identify plasmids is needed. This paper reports an improved nanofluidic optical mapping strategy to identify plasmids and locate the antibiotic resistance genes via the Cas9/CRISPR technique. We used the assay to analyze clinical samples from a potential nosocomial outbreak in an Ethiopian hospital, tracing the transmission routes among wards