Antibiotic resistance is a major threat to human health. Clinical situations are the main focus for antibiotic resistance research, but understanding the spread of resistance in the environment is also vital. A major contributor to this spread is wastewater from combined sewer overflow (CSO) events. The effect of CSO events on antibiotic resistance in the James River near Richmond, Virginia was studied using genomic and microbiological approaches. The abundance of genes associated with resistance to quinolones (qnrA) and tetracycline (tetW) was strongly correlated with the presence of fecal indicator bacteria (E. coli abundance) as well as total nitrogen and phosphorus loads, which suggests an anthropogenic source of these genes. Abundance of the blaTEM gene, which confers resistance to β-lactam antibiotics, was elevated during CSO events and increased with precipitation and river discharge. Bacteria isolated during a CSO event were resistant to more antibiotics and had higher multi-drug resistance when compared to isolates from a non-event. This study demonstrated that CSO events are contributing to the spread of antibiotic resistance.

<p>Objective: To explore views of medical doctors, veterinarians and drug sellers on use of antibiotics on humans and nonhumans and on factors that influences the development of resistance to antibacterial agents. Further, to look at the bi-directional relationship between antibiotic use and environment. </p><p>Methods: The study was a qualitative explorative interview study, analysed using conventional content analysis. It was conducted in Orissa, India. Data were collected by face to face semi structured interview. The interviews were tape recorded and transcribed into Oriya, then translated in to English. Each paragraph or sentence was coded. Similar codes were clustered together and collapsed into sub categories and categories. The main themes were allowed to emerge, based on the relationship between categories.</p><p>Findings: The main finding of the study was mishandling and abuse of antibiotics in patients as well as at professional level due to weak implementation of legislation, which appears to be the major cause of antibacterial agent resistance. Incomplete course or dose due to poverty in rural area and self medication in urban area are more common. The study also showed that climatic factors, pollution and population density are the major ecological factors which influence antibiotic prescriptions. Another major finding of this study was that, due to improper disposal system of pharmaceuticals; antibiotics are contaminating air, water and terrains which can cause major risk to aquatic and grazing animals. </p><p>Conclusion: This study emphasises the need for comprehensive actions including information, training, legislation and education at all levels of drug delivery system to rationalize antibiotic use by improving prescribing pattern and creating awareness among consumers. Proper disposal of pharmaceutical wastes is required to prevent the contamination of environment from pharmaceutical pollutants. Further study is essential concerning environmental impact of antibiotics.</p><p>Key words: Antibiotic use; antibiotic resistance; environment; qualitative; conventional; content analysis; veterinarians; medical doctors; drug sellers; Orissa; India.</p>

Plants, more precisely their chloroplasts with their bacterial-like expression machinery inherited from their cyanobacterial ancestors, can potentially offer a cheap expression system for proteinaceous pharmaceuticals. This system would be easily scalable and provides appropriate safety due to chloroplasts maternal inheritance. In this work, it was shown that three phage lytic enzymes (Pal, Cpl-1 and PlyGBS) could be successfully expressed at very high levels and with high stability in tobacco chloroplasts. PlyGBS expression reached an amount of foreign protein accumulation (> 70% TSP) that has never been obtained before. Although the high expression levels of PlyGBS caused a pale green phenotype with retarded growth, presumably due to exhaustion of plastid protein synthesis capacity, development and seed production were not impaired under greenhouse conditions. Since Pal and Cpl-1 showed toxic effects when expressed in E. coli, a special plastid transformation vector (pTox) was constructed to allow DNA amplification in bacteria. The construction of the pTox transformation vector allowing a recombinase-mediated deletion of an E. coli transcription block in the chloroplast, leading to an increase of foreign protein accumulation to up to 40% of TSP for Pal and 20% of TSP for Cpl-1. High dose-dependent bactericidal efficiency was shown for all three plant-derived lytic enzymes using their pathogenic target bacteria S. pyogenes and S. pneumoniae. Confirmation of specificity was obtained for the endotoxic proteins Pal and Cpl-1 by application to E. coli cultures. These results establish tobacco chloroplasts as a new cost-efficient and convenient production platform for phage lytic enzymes and address the greatest obstacle for clinical application. The present study is the first report of lysin production in a non-bacterial system. The properties of chloroplast-produced lysins described in this work, their stability, high accumulation rate and biological activity make them highly attractive candidates for future antibiotics. / Lytische Enzyme aus Bakteriophagen bieten Eigenschaften, die sie zu vielversprechenden Medikamenten im Einsatz gegen bakterielle Krankheiten machen. Obwohl sie speziell beim Einsatz gegen bakterielle Infektionen, welche durch Antibiotika resistente Erreger hervorgerufen werden, eine maßgebende Rolle spielen könnten, waren bisher die hohen Produktionskosten ein Hindernis für die medizinische Anwendung. Ein kostengünstiges und einfach zu handhabendes System, wie beispielsweise Chloroplasten in Pflanzen, würde diese lytischen Enzyme zu einer effizienten Alternative zu herkömmlichen Antibiotika machen. In dieser Arbeit wird erstmals die erfolgreiche Produktion von lytischen Enzymen in Tabak-Chloroplasten vorgestellt, welche mit einem Fremdproteingehalt von mehr als 70% des gesamtlöslichen Proteins der Pflanze eine Menge beschreibt, die bisher mit diesem Verfahren noch nicht erreicht wurde. Alle in Chloroplasten hergestellten lytischen Enzyme zeigten hohe spezifische bakteriolytische Aktivität gegen die gewählten Humanpathogene und waren innerhalb von Minuten in der Lage diese Bakterien abzutöten. Zur Herstellung von zwei lytischen Enzymen wurde in dieser Arbeit ein spezieller Shuttle-Vektor entworfen, der die Expression von toxischen Genen innerhalb von E. coli Zellen im Zuge der DNA Replikation vermeidet, jedoch die Herstellung einer ungehinderten Expression der toxischen Gene in den Chloroplasten nach Beseitigung des Selektionsmarkers erlaubte. Ein Vergleich zwischen einem herkömmlich verwendeten Transformationsvektor und dem Shuttle-Vektor mittels eines Reportergens zeigte, dass das neu entwickelte System bis zu 4 mal mehr Protein produzierte. Diese Ergebnisse zeigen das Potential von Chloroplasten als kostengünstige und leicht zu handhabende Produktionsplattform für lytische Enzyme, welche als neue Generation von Antibiotika attraktive Alternativen zu herkömmlichen Therapien bieten.

Objective: To explore views of medical doctors, veterinarians and drug sellers on use of antibiotics on humans and nonhumans and on factors that influences the development of resistance to antibacterial agents. Further, to look at the bi-directional relationship between antibiotic use and environment. Methods: The study was a qualitative explorative interview study, analysed using conventional content analysis. It was conducted in Orissa, India. Data were collected by face to face semi structured interview. The interviews were tape recorded and transcribed into Oriya, then translated in to English. Each paragraph or sentence was coded. Similar codes were clustered together and collapsed into sub categories and categories. The main themes were allowed to emerge, based on the relationship between categories. Findings: The main finding of the study was mishandling and abuse of antibiotics in patients as well as at professional level due to weak implementation of legislation, which appears to be the major cause of antibacterial agent resistance. Incomplete course or dose due to poverty in rural area and self medication in urban area are more common. The study also showed that climatic factors, pollution and population density are the major ecological factors which influence antibiotic prescriptions. Another major finding of this study was that, due to improper disposal system of pharmaceuticals; antibiotics are contaminating air, water and terrains which can cause major risk to aquatic and grazing animals. Conclusion: This study emphasises the need for comprehensive actions including information, training, legislation and education at all levels of drug delivery system to rationalize antibiotic use by improving prescribing pattern and creating awareness among consumers. Proper disposal of pharmaceutical wastes is required to prevent the contamination of environment from pharmaceutical pollutants. Further study is essential concerning environmental impact of antibiotics. Key words: Antibiotic use; antibiotic resistance; environment; qualitative; conventional; content analysis; veterinarians; medical doctors; drug sellers; Orissa; India.

I lactobacilli sono considerati dei microorganismi non-patogeni. Molti di loro appartengono al gruppo batterico GRAS e/o sono nell’elenco QPS. Dal momento che i lactobacilli intenzionalmente aggiunti agli alimenti possono agire come reservoir di geni di resistenza, la valutazione del rischio deve essere continuamente aggiornata. Lo scopo di questa tesi era la valutazione di alcuni metodi usati per testare e caratterizzare le specie del genere Lactobacillus per quanto riguarda la sicurezza e la potenziale attività probiotica. Nella prima parte due metodi di micro diluzione, il metodo ISO e CLSI, soni stati comparati testando la resistenza agli antibiotici di 54 ceppi L. plantarum. Sulla base di risultati ottenuti il metodo ISO era più adatto per valutare la resistenza di questa specie. Il test del limite di sensibilità della PCR per 8 paia di primers specifici per il rilevamento dei lactobacilli e bifido batteri da feci ha confermato i loro diversi livelli di efficacia. La seconda parte della tesi descrive un progetto di ricerca mirato sulla identificazione di nuovi ceppi probiotici fra diversi ceppi di Lactobacillus paracasei e Lactobacillus rhamnosus identificando dei geni o loci responsabili della interazione con l’ospite, immunomodulazione, e l’inibizione della crescita dei patogeni. Le analisi fenotipiche dei 40 ceppi hanno confemato una grande variabilità fra di loro, che può servire per associare delle caratteristiche fenotipiche a quelle genotipiche. Tra i ceppi dello stesso progetto è stato individuato un ceppo di L. mucosae. Dal momento che questa è una specie relativamente nuova, le sue caratteristiche sono state analizzate comparandole con altri 3 ceppi appartenenti alla stessa specie. In questo modo sono state confermate alcune informazioni su L. mucosae, ma soprattutto sono stati forniti dei dati nuovi sulle proprietà di questa specie. / The species of the Lactobacillus genus are generally believed to be microorganisms with no pathogenic potential. Many of them have granted GRAS and QPS status. Non-pathogenic bacteria as lactobacilli-intentionally added or accidentally present in food-are under evaluation, as they could act as reservoir of resistant genes. This thesis was aimed to evaluate some methods used for testing and to characterize some Lactobacillus species, as regards their safety and potential probiotic activity. The first part of the research focused on the comparison of two broth microdilution methods: ISO and CLSI, in order to assess the resistance of 54 L. plantarum strains to antimicrobial agents. The results suggest better performances of the phenotypic assay developed by ISO, at least for strains belonging to L. plantarum species.Then the assessment of the PCR detection limit for 8 sets of primers for the detection of lactobacilli and bifidobacteria from infant faeces confirmed different levels of effectiveness for the primers. Next part of the thesis was the research project aimed at identifying genes or genetic loci of different strains of two Lactobacillus species (i.e. Lactobacillus paracasei and Lactobacillus rhamnosus) involved in the interaction with the host, immune-modulation of host cells and pathogen growth inhibition in order to find new probiotic strains. The phenotypic analysis of 40 selected strains demonstrated large variability between strains of these species, which could serve to the association of phenotypic differences to genome specificities. A strain of Lactobacillus mucosae was found within the framework of the same project. As it is a relatively new species, it was chosen to further investigate its properties, comparing it with three other L. mucosae strains. This study led to confirm some information but first and foremost it has provided new data on the examined species.

The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria. In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection. These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.

Contexte : les règlements distinguent habituellement les médicaments prescrits uniquement (prescription only medicines POM) et les médicaments en vente libre (over-the-counter OTC). Les premiers nécessitent une prescription médicale et les derniers sont disponibles pour l'automédication sans ordonnance. Au Liban comme dans les autres pays de l’Est de la Méditerranée, les médicaments théoriquement prescrits peuvent facilement être achetés sans ordonnance. Les antibiotiques sont parmi les classes de médicaments les plus fréquemment vendus sans ordonnance. L'utilisation inappropriée d'antibiotiques augmente le taux de résistance bactérienne et la fréquence de l'échec du traitement. Cette étude est menée pour déterminer le mode d'emploi des antibiotiques disponibles en tant qu'automédication au Liban, en comparaison avec leur utilisation comme médicaments uniquement prescrits en France. Méthode : Tout d'abord, une revue approfondie de la littérature publiée (1990-2015) a été menée en utilisant les bases de données : Pubmed, Web of Science, Cochrane et Google Scholar, pour abus de médicamentsdisponibles sans ordonnance au Moyen-Orient ;les antibiotiques étaient parmi les médicaments les plus mal utilisés. Une étude prospective a ensuite été menée au Liban. Les données ont été collectées auprès de patients qui achetaient des antibiotiques avec ou sans ordonnance dans les officines sur une période d’un an (septembre 2015 à septembre 2016). En France, les données ont été extraites de la base de données Echantillon Généraliste de Bénéficiaires (EGB), contenant un échantillon représentatif permanent 1/97 du système d'assurance-maladie national français, qui contient toutes les prescriptions dispensées en France. Nous y avons mené des recherchespour identifier les modes d'utilisation des 5 antibiotiques les plus utilisés au Liban. Résultats : La distribution moyenne de ces antibiotiques était plus élevée en France (18,6 DDD) qu'au Liban (10.6DDD). Cependant, le nombre moyen de dérogations au Liban était de 3,39 par an, plus élevé qu'en France (2 par an). Ce n'était pas différent entre les participants avec ou sans ordonnance. Le coamoxiclav a été largement consommé au Liban à la fois sous ordonnance et par automédication, alors que l'amoxicilline était le principal antibiotique consommé en France.62,4% des patients ont utilisé le bon antibiotique et 80,1% l'ont utilisé dans le dosage correct. Dans la suivi, la durée du traitement était inappropriée dans la majorité des cas (68,6%). Lorsque tous ces trois facteurs ont été additionnés, il s'avère que 83,6% des antibiotiques ont été utilisés de manière inappropriée. La pertinence de l'utilisation a été observée chez 27,6% et 16,4% des antibiotiques prescrits et non prescrits, respectivement. Conclusion : Il est certain que l'automédication observée ou l'utilisation médicalement non surveillée d'antibiotiques est un risque majeur. Les premières mesures à prendre pour réduire celles-ci sont éducatives, pour les patients, les pharmaciens, les prescripteurs, l'industrie pharmaceutique et les autorités réglementaires. / Background: Regulations usually distinguish between prescription-only (POM) and over-the-counter (OTC) medicines. The former requires medical prescription; the latter are available for self-medication (SM) without a prescription. In Lebanon as in other Eastern Mediterranean countries, theoretically prescription medicines can easily be purchased without a prescription. Antibiotics are among the most commonly sold drug classes without prescription. The inappropriate use of antibiotics increases the rate of bacterial resistance and the frequency of treatment failure. This study is conducted to determine the usage pattern of antibiotics available as self-medication in Lebanon, compared with their use as prescription only drugs in France. Method: First, an extensive review of the published literature (1990–2015) was conducted using Pubmed, Web of Science, Cochrane and Google Scholar databases, for OTC medicine misuse and abuse in the Middle East. Antibiotics were among the most commonly misused medications. A cross sectional prospective field study was then conducted in Lebanon. Data was collected from patients seeking antibiotics with or without prescription in community pharmacies over a 1-year period (September 2015 to September 2016). In France, data was extracted from the EchantillonGénéraliste de Bénéficiaires (EGB) database, the permanent 1/97 representative sample from the French national healthcare insurance system which contains all dispensed prescriptions in France. It was searched in 2013 to identify usage patterns of antibiotics, of 5 antibiotics most commonly used in Lebanon. Results: The average dispensing for these antibiotics was higher in France (18.6 DDD) than that in Lebanon (10.6DDD). However, the average number of dispensings in Lebanon was 3.39 per year, higher than that in France (2 per year). It was not different between participants with or without prescription. Coamoxiclav was widely consumed in Lebanon as both prescription and self-medication, whereas amoxicillin was the main antibiotic in France. 62.4% of patients used the right antibiotic and 80.1% used it in correct dosage. The duration of treatment in the follow up was inappropriate in the majority of cases (68.6%). When all of these three factors were summed together, it turned out that 83.6% of antibiotics were utilized inappropriately. Appropriateness in use was seen in 27.6% and 16.4% of the prescribed and non-prescribed antibiotics respectively. Conclusion: Misuse of antibiotics is a major driver in the development of antibiotic resistance, an issue for individuals and for public health. Obviously the observed self-medication or medically unsupervised utilization of antibiotics is a major risk. The first measures to take to reduce this are educational, for patients, pharmacists, prescribers, the pharmaceutical industry and the regulatory authorities.

Methicillin-resistant Staphylococcus aureus (MRSA) is a growing concern in Canada especially in Aboriginal communities in remote regions. The northern and remote communities possess some or many of the risk factors that are identified in previous research to be associated with Community-Associated Methicillin-resistant Staphylococcus aureus (CA-MRSA) infections such as overcrowding, challenges in maintaining personal hygiene and limited access to healthcare. CA-MRSA spreads rapidly in the communities. It is known to not only affect young and healthy individuals, but it is also associated with high morbidity and mortality rates. Furthermore, antibiotic resistance in CA-MRSA is increasing in Canada. It is known that inappropriate and greater use of antibiotics is associated with increased antibiotic resistance. Resistant CA-MRSA infections are more difficult to treat. To reduce and to control the antibiotic resistance, monitoring the prevalence of CA-MRSA and the changing antibiotic susceptibility profiles at the population level, especially in highly affected communities, is crucial. By monitoring this trend, it will enable healthcare practitioners to provide more adequate and appropriate treatment. To our knowledge, there was no community-based study that examined the epidemiology of CA-MRSA skin and soft-tissue infections (SSTIs) in First Nations communities in Canada at large scale, and the knowledge on the risk factors, outcomes and antibiotic susceptibility profiles is still very limited. This study aimed to describe the local epidemiology of SSTIs at the community level in selected First Nations communities as well as to describe the antibiotic use to treat SSTIs and the antibiotic susceptibility patterns of CA-MRSA. A retrospective chart review was conducted in 12 nursing stations in the First Nations communities across 5 provinces in Canada. The charts of individuals over 18 years of age who had received service at the nursing station in the previous 12 months, starting retrospectively from the date of collection, were reviewed in this study. Each antibiotic prescription that was noted in the chart in this period prior to chart review was recorded in the antibiotic tracking case report form. Data collected included demographics, indication for antibiotic use, antibiotic prescription parameters and patient outcomes. In total, of 372 patient charts reviewed, 224 patient charts contained at least one case (an encounter that resulted in an antibiotic prescription during the study period). Of those 224 charts, 459 cases were recorded and, of those, 137 cases had a diagnosis of an SSTI. In the 65 patients accounting for all cases of SSTI patients, more than 80% of the study population were under the age of 50. The prevalence of impaired renal function was low, diabetes was present in 20% of cases, cardiac disease was present in 15% of cases, and reported alcohol misuse was present in 30% of cases. The presence of indwelling devices was very rare. There were 137 cases of SSTIs over 372 charts reviewed in total. The prevalence of SSTI among the selected First Nations communities in 2012-2013 was estimated at almost 37% (137 cases of SSTIs / 372 charts reviewed). In the 137 SSTI cases, 55 cases were identified as MRSA infections either by laboratory test such as wound culture or by history of colonization documented in the chart. The overall prevalence of MRSA in all SSTI cases was estimated at 40.1% (55 confirmed MRSA positive cases / 137 cases of SSTIs). The majority of SSTIs were purulent infections and wounds. We also found that a wound culture and susceptibility test were performed only in 29% of all SSTI cases. An orally administered antibiotic was most frequently used (in 71.5% of treatments). Topical antibiotics were used in 18.3% and IV antibiotics were used in 8.8%. Other than the antibiotic treatment, wound care was performed in 49% of the SSTI cases and incision and drainage (I&D) procedure in 9%. The majority of MRSA isolates in this study were susceptible to clindamycin and co-trimoxazole (90.5% and 95.2%), but only 29% were susceptible to erythromycin. In general, higher rates of SSTIs were seen in communities where overcrowding and poor access to running water are more prevalent. In this study, we found that the diagnostic tests such as wound culture and sensitivity test was not ordered very often and there was also lack of follow-up or lack of documentation of the follow-up. More research is needed to better understand some of the challenges and risk factors associated with CA-MRSA infections in remote communities. Developing a national-level surveillance system that can help with monitoring the epidemiology of SSTIs and the antibiotic susceptibility test results of CA-MRSA at community level would be essential for better prevention, control, and management. Furthermore, adopting other initiatives such as antibiotic stewardship programs at community and healthcare settings as well as addressing the socio-environmental factors such as housing and access to water would be all very important in the steps to curb antibiotic resistance.

In a world where the human population is increasing, new innovations to produce enough food are required. Aquaculture’s part of the global animal protein production has increased in recent years and could be a possible solution. However, if aquaculture is poorly managed, it can result in negative consequences and one such consequence is the development of antibiotic resistance. In this review, I examine how aquaculture affect antibiotic resistance by studying what the literature says on accumulation of antibiotics in different organisms and sediment, if antibiotics can be transferred to humans through consumption of antibiotic treated products, and if human pathogens in aquaculture farms may acquire antibiotic resistance. Furthermore, I examine what factors are contributing to irresponsible antibiotic use and how such use is managed. The result of this review indicate that antibiotics are able to accumulate in organisms and sediment. It is not clear however how consumption of these affect human microbiomes. In contrast, it is clear that antibiotic resistance can be transferred from antibiotic-resistant bacteria to human pathogens. Regarding antibiotic use, irresponsible use foremost exists in low-income countries and the main drivers behind such use are socioeconomic ones, such as lack of knowledge, poverty and food security. Finally, I propose possible solutions that might improve future management. / I en värld där den mänskliga befolkningen ökar krävs nya innovationer för att producera tillräckligt med mat. Vattenbrukets andel av den globala animaliska proteinproduktionen har ökat de senaste åren och kan ses som en potentiell lösning. Om vattenbruk dock hanteras ansvarslöst kan det uppstå negativa konsekvenser. En sådan konsekvens är utveckling av antibiotikaresistens hos skadliga bakterier. I denna litteraturstudie undersöker jag vattenbrukets påverkan på antibiotikaresistens genom att studera vad litteraturen säger om ackumulation av antibiotika i olika organismer och sediment, om antibiotika kan överföras till människor genom konsumtion av antibiotikabehandlade produkter, och om mänskliga patogener i vattenbruksodlingar kan förvärva antibiotikaresistens. Jag undersöker också vilka faktorer som bidrar till ansvarslös antibiotikaanvändning och hur den hanteras ur ett hållbarhetsperspektiv. Resultaten i denna studie tyder på att antibiotika kan ackumuleras i organismer och sediment men att det råder oklarheter huruvida konsumtion av antibiotikabehandlad mat påverkar mänskliga bakteriekulturer. Antibiotikaresistens kan dock överföras från antibiotikaresistenta bakterier till mänskliga patogener. Ansvarslös antibiotikaanvändning sker huvudsakligen i fattigare länder och det är förmodligen i stor utsträckning till följd av socioekonomiska faktorer som okunskap, fattigdom och livsmedelstrygghet. Slutligen föreslår jag lösningar som möjligen kan bidra till bättre hantering av framtida antibiotikaanvändning.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22762号 / 工博第4761号 / 新制||工||1745(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 米田 稔, 准教授 松田 知成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM