Single DNA Molecule Analysis – New Tools for Medical Diagnosis

The DNA molecule, the blueprint of life, contains an enormous amount of information. The information is coded by the combination of four bases; adenine, cytosine, guanine, and thymine, that, together with the sugar-phosphate backbones, make up the DNA double helix. There are variants in the human DNA sequence that are related to the onset and progression of disease. Under different conditions the DNA can also be damaged, which if not repaired correctly can result in a shortened life span, rapid ageing and development or progression of a variety of diseases, including cancer. Human disease can also be induced by external factors in our surroundings, such as pathogens. One of the cornerstones in modern medicine has been the use of antibiotics to prevent and treat these pathogenic infections, but the global spread of antibiotic resistance is today one of the largest threats to mankind according to the World Health Organization. One consequence of a large global increase in antibiotic resistance would be that routine surgery or chemotherapy treatment might be considered too perilous, because there are no drugs available to prevent or treat the bacterial infections that are closely connected with these procedures.Novel techniques are needed to characterize different features of DNA in medicine and diagnostics. Single molecule analysis is one method to unveil different kinds of information from individual biomolecules, such as DNA. This thesis uses fluorescence microscopy to shine light upon such information in single DNA molecules from both humans and bacteria, and with that unveil important biological and medical characteristics of that DNA. It describes one method for identifying and quantifying DNA damage induced by a chemotherapy agent, helping to understanding the processes of DNA damage and repair related to diseases and medical treatments. Another method developed is for rapid identification of bacterial infections, with the classification of bacterial sub-species groups and identification of antibiotic resistance genes on plasmids. The methods have the potential to rapidly provide comprehensive diagnostics information, to optimize either early antibiotic treatment or chemotherapy treatment, and thereby enable future precision medicine management

Strain-level bacterial typing directly from patient samples using optical DNA mapping

For bacterial infections, it is important to rapidly and accurately identify and characterize the type of bacteria involved so that optimal antibiotic treatment can be given quickly to the patient. However, current diagnostic methods are sometimes slow and cannot be used for mixtures of bacteria. We have, therefore, developed a method to identify bacteria directly from patient samples. The method was tested on two common species of disease-causing bacteria - Escherichia coli and Klebsiella pneumoniae - and it could correctly identify the bacterial strain or subtype in both urine samples and mixtures. Hence, the method has the potential to provide fast diagnostic information for choosing the most suited antibiotic, thereby reducing the risk of death and suffering. 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. BackgroundIdentification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.MethodsIn this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections.ResultsWe demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids.ConclusionThe optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management

Development of alkane-induced biosensor in Saccharomyces cerevisiae

Den ökande halten av växthusgaser i atmosfären kräver en utveckling av nya miljövänliga bränslen. Användning av mikrober vid bränsletillverkning är en lovande metod som har varit aktuell i flera år. n-Alkaner har en hög likhet med bland annat diesel och kan därför direkt ersätta nuvarande fossila bränslen. Stammar av Saccharomyces cervisiae som kan producera n-alkaner har tagits fram. Dessa stammar är långt ifrån optimala och behöver utvecklas innan de kan nyttjas på industriell skala. Målet med detta projekt är att skapa en biosensor som kan användas som ett screeningverktyg för att optimera utbytet av n-alkaner i S. ce- revisiae. Ett transkriptionssystem från Yarrowia lipolytica tillsammans med GFP används för att skapa biosensorn. Systemet från Y. lipolytica består av tre gener, YAS1, YAS2 och YAS3, samt promotorsekvensen ARE1. Detta systemet aktiverar transkription i närvaro av n-alkaner. PCR-produkter genererades med framgång och användes för att konstruera plasmider innehållande kombinationer av YAS1, YAS2, YAS3, ARE1-sekvenser samt GFP. Veri_kation av de konstruerade plasmiderna bekräftade att generna inkorporerats med få mutationer. Biosensorn introducerades i S. cerevisiae med ett två-vektorsystem. Flödescytometri användes för att testa biosensorn i närvaro och frånvaro av dekan och en skillnad i uttryck bekräftades. Resultaten indikerar på att det är möjligt att implementera systemet från Y. lipolytica i S. cerevisiae med målet att skapa en biosensor för n-alkane

CRISPR/CAS9 BASED DNA-COMBING ASSAY FOR DETECTING ANTIMICROBIAL RESISTANCE GENES ON PLASMIDS

We present a method based on CRISPR/Cas9 excision and DNA combing to detect anti-microbial resistance (AMR) genes on bacterial plasmids. The assay is inexpensive, simple, fast, and also provides information on the number and size of plasmids in a sample. We demonstrate detection of the gene encoding for the New Delhi metallobeta-lactamase 1 (blaNDM-1) enzyme, known to make bacteria resistant to a broad range of beta-lactam antibiotics

CRISPR/CAS9 BASED DNA-COMBING ASSAY FOR DETECTING ANTIMICROBIAL RESISTANCE GENES ON PLASMIDS

We present a method based on CRISPR/Cas9 excision and DNA combing to detect anti-microbial resistance (AMR) genes on bacterial plasmids. The assay is inexpensive, simple, fast, and also provides information on the number and size of plasmids in a sample. We demonstrate detection of the gene encoding for the New Delhi metallobeta-lactamase 1 (blaNDM-1) enzyme, known to make bacteria resistant to a broad range of beta-lactam antibiotics

Bacterial identification by optical mapping of genomic DNA in nanofluidic channels

A variety of pathogenic bacteria can infect humans and the increase in bacteria resistant to common antibiotics is a large threat to human health worldwide. This work presents a method, based on optical DNA mapping (ODM) in nanofluidic channels, that can detect the type of bacterial present in a sample by matching the obtained maps of large DNA molecules to a database of fully assembled bacterial genomes. The extraction and labelling protocol has been designed to work for both Gram-positive and Gram-negative bacteria, not requiring any prior knowledge about the sample content

Cultivation-Free Typing of Bacteria Using Optical DNA Mapping

A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often be time-consuming and depend on the cultivation of the bacterial pathogen(s). Here, we present a stand-alone, enzyme-free, optical DNA mapping assay capable of species identification by matching the intensity profiles of large DNA molecules to a database of fully assembled bacterial genomes (>10 000). The assay includes a new data analysis strategy as well as a general DNA extraction protocol for both Gram-negative and Gram-positive bacteria. We demonstrate that the assay is capable of identifying bacteria directly from uncultured clinical urine samples, as well as in mixtures, with the potential to be discriminative even at the subspecies level. We foresee that the assay has applications both within research laboratories and in clinical settings, where the time-consuming step of cultivation can be minimized or even completely avoided

Bacterial identification by optical mapping of genomic DNA in nanofluidic channels

A variety of pathogenic bacteria can infect humans and the increase in bacteria resistant to common antibiotics is a large threat to human health worldwide. This work presents a method, based on optical DNA mapping (ODM) in nanofluidic channels, that can detect the type of bacterial present in a sample by matching the obtained maps of large DNA molecules to a database of fully assembled bacterial genomes. The extraction and labelling protocol has been designed to work for both Gram-positive and Gram-negative bacteria, not requiring any prior knowledge about the sample content

A simple cut and stretch assay to detect antimicrobial resistance genes on bacterial plasmids by single-molecule fluorescence microscopy

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

AMP-activated protein kinase and metabolic control.

28 pagesInternational audienceAMP-activated protein kinase AMP-activated protein kinase (AMPK AMPK ), a phylogenetically conserved serine/threonine protein kinase, is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. It is now recognized that pharmacological activation of AMPK improves blood glucose homeostasis, lipid profile, and blood pressure in insulin-resistant rodents. Indeed, AMPK activation mimics the beneficial effects of physical activity or those of calorie restriction calorie restriction by acting on multiple cellular targets. In addition, it is now demonstrated that AMPK is one of the probable (albeit indirect) targets of major antidiabetic drugs drugs including the biguanides (metformin metformin ) and thiazolidinedione thiazolidinedione s, as well as of insulin-sensitizing adipokines (e.g., adiponectin adiponectin ). Taken together, such findings highlight the logic underlying the concept of targeting the AMPK pathway for the treatment of metabolic syndrome and type 2 diabetes