The prevalence of antibiotics on farms for therapeutic and prophylactic use in animals can cause negative effects on biomethane production during anaerobic digestion. Previous literature has found decreased biomethane production rates from a variety of antibiotics, but biogas inhibition differs between studies of continuous and batch reactors and the type of antibiotic studied. Cephalosporin drugs are the most common antibiotic class used to treat mastitis in dairy cows and can retain most of their bioactivity after excretion. Ceftiofur is a commonly used cephalosporin drug but no previous study investigating the effect of Ceftiofur on biomethane during continuous anaerobic digestion has been performed. The aim of this study was to examine the effect on biomethane production when manure from cows treated with Ceftiofur was anaerobically digested. Laboratory sized anaerobic digesters (AD) were run at thermophilic (55°C) temperatures and a 10 day hydraulic retention time. Manure from cows treated with Ceftiofur were fed to the antibiotic treatment reactors for 50 days. The reactor performance was measured by i) biomethane production, ii) waste stabilization in terms of solids and chemical oxygen demand, iii) change in mass of Ceftiofur and iv) change in concentration of antibiotic resistant genes, specifically cfx(A), mef(A), and tet(Q). There was statistically significant decrease in cumulative gas production due to the addition of Ceftiofur into the reactors, but no significant difference between treatments in waste stabilization in terms of percent volatile solids (VS) and total chemical oxygen demand (TCOD) reduction. Anaerobic digestion decreased the amount of Ceftiofur in manure, and the amount of Ceftiofur in the reactors reduced over the time of the experiment. Change in antibiotic resistant genes (ARGs) were gene dependent over time. Concentrations of tet(Q) reduced significantly between feed and effluent of both treatments, and cfx(A) reduced significantly for the control treatment but not the Ceftiofur treatment. Concentrations of mef(A) increased over time in both treatments. Overall, the addition of Ceftiofur in continuously operated anaerobic digesters negatively affected biomethane production, a value-added product responsible for on-farm renewable energy. However, anaerobic digestion does decrease the mass of Ceftiofur within manure, thereby reducing the environmental loading from run-off from farms. / Master of Science

京都大学 / 新制・課程博士 / 博士(工学) / 甲第25269号 / 工博第5228号 / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 西村 文武, 教授 米田 稔, 教授 松田 知成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

Due to the recent rise in antibiotic resistant pathogens, and the difficulties surrounding the quest for new antibiotics, many researchers have started revisiting antibiotic interactions in hopes of finding new treatment options. The primary outcome of this project was to examine the efficacy of concomitant antibiotic use under varying nutrient conditions, to identify variations in antibiotic interactions. Antibiotic interactions were studied, utilizing E. coli as a model bacterial system, grown in four different media types. E. coli cultures were treated with streptomycin, tobramycin, erythromycin, and amikacin individually and in a pairwise fashion at varying doses. We found that at least some antibiotic efficacies were dependent on the environmental nutrient conditions E. coli was grown in, as the antibiotics were not equally effective in all media types. E. coli grown in potato dextrose broth, in particular, showed extremely high tolerance to antibiotic inhibition. In addition, we observed several variations in antibiotic interactions, depending on the combination of antibiotics and environmental conditions utilized. It is predicted that differences in available nutrients is the primary cause of the observed discrepancies in antibiotic properties between media. The observation of changes in antibiotic efficacy under different environmental and nutrient conditions has serious implications for use of antibiotic combinations as drug treatments. Not all microenvironments within the human body have identical nutrient make-up. If the interactions antibiotics are reported to have in one environmental condition change under another, reckless prescription of combinations could lead to a serious adverse reaction. Thus, this is an important area for future in vitro and in vivo research.

In evolution alternative genetic trajectories can potentially lead to similar phenotypic outcomes. However, certain trajectories are preferred over others. These preferences bias the genomes of living organisms and the underlying processes can be observed in ongoing evolution. We have studied a variety of biases that can be found in bacterial chromosomes and determined the selective causes and functional consequences for the cell. We have quantified codon usage bias in highly expressed genes and shown that it is selected to optimise translational speed. We further demonstrated that the resulting differences in decoding speed can be used to regulate gene expression, and that the use of ‘non-optimal’ codons can be detrimental to reading frame maintenance. Biased gene location on the chromosome favours recombination between genes within gene families and leads to co-evolution. We have shown that such recombinational events can protect these gene families from inactivation by mobile genetic elements, and that chromosome organization can be selectively maintained because inversions can lead to the formation of unstable hybrid operons. We have used the development of antibiotic resistance to study how different bacterial lifestyles influence evolutionary trajectories. For this we used two distinct pairs of antibiotics and disease-causing bacteria, namely (i) Mycobacterium tuberculosis that is treated with rifampicin and (ii) Escherichia coli that is treated with ciprofloxacin. We have shown that in the slow-growing Mycobacterium tuberculosis, resistance mutations are selected for high-level resistance. Fitness is initially less important, and over time fitness costs can be ameliorated by compensatory mutations. The need for rapid growth causes the selection of ciprofloxacin resistance in Escherichia coli not only to be selected on the basis of high-level resistance but also on high fitness. Compensatory evolution is therefore not required and is not observed. Taken together, our results show that the evolution of a phenotype is the product of multiple steps and that many factors influence which trajectory is the most likely to occur and be most beneficial. Over time, selection will favour this particular trajectory and lead to biased evolution, affecting genome sequence and organization.

Klebsiella pneumoniae is an opportunistic pathogen responsible for lobar pneumoniae, liver abscess, and septicemia. Clinical isolates are found to be extended spectrum beta lactamase positive with differential expression of the two classical porins, OmpK35 and OmpK36. Porin loss is associated with increased minimum inhibitory concentrations of beta lactam, cephalosporin, and carbapenem antibiotics that target the peptidoglycan. However, little is known about how porin loss affects other aspects of the cell envelope. The focus of this study was to characterize clinical isolates exhibiting differential porin expression and determine if the cumulative changes altered the resistance to phagocytosis by macrophages. The results support the hypothesis that porin loss significantly impacts the overall cell envelope composition, which in turn alters interactions with macrophages.

Pseudomonas aeruginosa et Stenotrophomonas maltophilia, pathogènes opportunistes majeurs, pourraient acquérir leur résistance aux antibiotiques dans l’environnement, sous la pression exercée par les métaux lourds par co-sélection de résistance. Nous avons tout d’abord évalué la distribution et l’abondance de ces espèces dans un large panel de sols d’origine géographique différente (France et Afrique) et évalué l’influence d’activités anthropiques susceptibles d’exposer les sols en éléments métalliques sur cette distribution. Alors que la présence de P. aeruginosa est sporadique et plutôt liée à un apport exogène, S. maltophilia est présente dans tous les sols étudiés, suggérant son endémicité. L’évaluation des résistances des souches isolées de ces sols a également montré des différences entre les deux espèces. Les souches environnementales de P. aeruginosa sont pour la plupart caractérisées par un phénotype sauvage alors que celles de S. maltophilia présentent une grande diversité de phénotypes en fonction des sites, parfois similaires à ceux de souches cliniques. Cette diversité peut être attribuée à l’adaptation aux conditions environnementales très différentes rencontrées mais il est difficile d‘attribuer précisément aux métaux un rôle dans la co-sélection de ces résistances. L’étude menée sur la communauté bactérienne d’un sol contaminé a également permis de mettre en évidence une forte proportion de bactéries résistantes à différents antibiotiques représentée par des espèces qualifiées de pathogènes opportunistes ainsi que la présence du gène blaIMP, permettant la résistance à l’imipénème, utilisé en milieu clinique pour le traitement de clones multi-résistants. / Pseudomonas aeruginosa and Stenotrophomonas maltophilia, two major opportunistic pathogens, could acquire antibiotic resistance in the environment under heavy metal pressure that co-selects both resistances. We first investigated the distribution and abundance of these species in a wide range of soils of different geographical origin (France and Africa) and evaluated the influence of human activities that may expose soils to metallic elements on this distribution. While the presence of P. aeruginosa is rather sporadic and could be linked to exogenous intake, S. maltophilia is present in all studied soils, that suggests its endemicity. Evaluating resistance capacities of strains isolated from these soils also showed differences between the two species. Environmental strains of P. aeruginosa are mostly characterized by a wild type phenotype, whereas those of S. maltophilia present a wide diversity of phenotypes depending on the site, sometimes similar to those of clinical strains. This diversity could be attributed to a deep adaptation to the very different environmental conditions encountered in the original niche but it is difficult to attribute specifically to metals a role in coselection of resistance. The study conducted on the bacterial community present in a contaminated soil has also highlighted a high proportion of bacteria resistant to different antibiotics represented by species qualified as opportunistic pathogens and the presence of the gene blaIMP, enabling resistance to imipenem, used in the hospital to treat infections due to multidrug-resistant clones.

Comprendre l'émergence et la propagation des maladies infectieuses chez les animaux sauvages est devenue une priorité en santé publique et en conservation. En combinant la collecte de données et le développement de modèles épidémiologiques, cette thèse s'est focalisée sur la compréhension de deux phénomènes clés: (i) étudier comment l'hétérogénéité au niveau des individus, des groupes, de la population et de l'environnement influence la propagation de parasites et (ii) quantifier la transmission de bactéries résistantes aux antibiotiques depuis l'homme vers les animaux sauvages dans trois aires protégées d'Afrique (Tsaobis NP- Namibie, Lopé NP-Gabon et Dzanga-Ndoki NP- République Centrafricaine). Les principaux résultats de ce travail montrent que : (1) De multiples facteurs incluant la température, la pluie, l'utilisation du territoire, le genre, l'âge et la condition physique influencent la richesse spécifique de parasites gastro-intestinaux chez le babouin chacma, (2) Les contacts entre animaux autour des points d'attractions de l'habitat peuvent influencer de manière importante la propagation spatio-temporelle d'une maladie, (3) La transmission d'entérobactéries résistantes semble avoir lieu depuis les humains ou le bétail vers les animaux sauvages dans des zones où le contact entre ces populations est élevé, (4) Le gradient de densité de gorilles produit par la chasse peut générer une augmentation de prévalence d'un parasite avec la distance au point d'introduction. Les conclusions de ce travail ouvrent de nouvelles possibilités pour l'étude des maladies émergentes chez les animaux sauvages. / Understanding the emergence and spread of infectious disease in wild animal populations has become an important priority for both public health and animal conservation. Combining the collection of empirical data with the development of epidemiological models, this thesis focuses on understanding two key issues of wildlife epidemiology: (i) how heterogeneity at the individual, group, population and landscape level affects parasite spread (ii) investigating whether transmission of antibiotic resistant bacteria from humans to wildlife is occurring within three protected areas of Africa (Tsaobis NP-Namibia, Lope NP-Gabon and Dzanga-Ndoki NP-Central African Republic). The main findings of this work indicated that: (1) multiple-scale factors including temperature, rainfall, home range use, sex, age and body condition influence gastro-intestinal parasite richness among wild baboons; (2) animal contacts around ‘habitat hotspots' can substantially influence the spatio-temporal dynamics of a disease; (3) antibiotic resistant enterobacteria seem to be spreading from humans/livestock to wildlife when the territory overlap between these two populations is expected to be high; (4) gradients in gorilla density created by bushmeat hunting can reverse the expected pattern of decreasing parasite prevalence with distance to human-spillover. The conclusions of this work open new possibilities for studying the mechanisms explaining the spread of emerging infectious diseases among wild animals.

I examensarbetet Det post-antibiotiska köket har samhällsfrågan om antibiotikaresistens undersökts ur ett designperspektiv. Detta för att uppmärksamma och sprida kunskap om problemet med den ökande resistensen och den nutida konsumtionen av läkemedlet. Designmetoden som använts är spekulativ design och syftet har varit att tillverka fem objekt som skall påverka och motivera en publik till att förändra den aktuella användningen av mirakelmedicinen. Genom grundliga efterforskningar om problematiken och om en framtid utan antibiotika har ett scenario formulerats. Detta scenario utspelar sig 30 år framåt i tiden, år 2049, i en värld där det inte längre finns fungerande antibiotika. Hur utförs en vardagsaktivitet, som att laga spagetti och köttfärssås, när ett litet sår kan leda till en dödlig infektion? Baserat på efterforskningar, workshops och samtal kunde köksobjekten fastställas; en kniv som minskar risken för stick- och skärskador, skyddande handskar, halkfria skor, en ansiktsmask och bakteriekryddor. Objekt som kan bli en del av det post-antibiotiska köket och vardagslivet om vi inte ändrar vårt nutida beteende. / In the degree work The Post-Antibiotic Kitchen the societal issue concerning antibiotic resistance has been explored through design. The purpose of the project has been to bring attention and spread knowledge about the growing resistance and current consumption of antibiotics. Speculative design has been the used design method and the goal has been to create five objects that will influence and motivate an audience to change the present usage of antibiotics. Through thorough research on the issue and on a future without antibiotics, a scenario has been formulated. The scenario takes place 30 years in the future, in 2049, in a world where there are no longer any functioning antibiotics. How will a day to day activity, such as cooking a meal, be performed when a small cut could lead to a deadly infection? Based on research, workshops and conversations five objects were created; a knife to prevent cut- and stab injuries, protective gloves, non-slip shoes, a face-guard and bacteria spices. Five objects that could be a part of the post-antibiotic kitchen and the daily life unless we change our current behavior today. / <p>Posten kompletterad 20190813 med uppdaterad version av uppsatsen.</p>

Urinvägsinfektioner är den näst vanligaste anledningen till antibiotikaförskrivning i Sverige. För diagnos av UVI i urinkultur med standardmetoden krävs 18 - 48 timmar och ytterligare 24 timmar i odling tills resultaten för antimikrobiell känslighetstestning är tillgängliga. Direktresistensmetoden ger svar 24 timmar tidigare än standardmetoden. Studiens syfte var att utvärdera direktresistensmetoden (in-housemetod) från mikrobiologiska laboratoriet Unilabs, Skövde på urinodlingar från patientprover och jämföra den med EUCAST-diskdiffusionsmetod. I denna studie användes 334 bakteriestammar från urinodlingar; E. coli (n=211) E. faecalis (n=45), Klebsiella spp (n=37), P. mirabilis (n= 17) och övriga gramnegativa bakteriestammar (n=24). Direktresistensmetoden och EUCAST diskdiffusionsmetod har utförts parallellt på alla bakteriestammar och sex olika antibiotikatyper testades. Hämningszonernas diameter lästes av och placerades i rätt S, I, R kategori för respektive antibiotikum. Från sammanlagt 1674 testade antibiotikalappar hittades 23 avvikelser där E. coli representerade 13 av dessa. Mecillinam var det antibiotikum där flest avvikelser noterades, totalt tio stycken. Överensstämmelsen mellan de två metoderna var 98,6% från 1674 testade antibiotikalappar. Direktresistensmetoden leder till att patienten få ett svar 24 timmar tidigare än med EUCAST diskdiffusionsmetoden där svaret inkluderar även resistensbestämning. / Urinary tract infections are the second most common reason for prescription of antibiotics in Sweden. Diagnosis of UTIs in urine culture by the standard method requires 18 to 48 hours and an additional 24 hours until antimicrobial testing is available. The direct resistance method provides answers 24 hours earlier than the standard method. The aim of the study was to evaluate the direct resistance method (in-house method) from the microbiological laboratory Unilabs, Skövde on urine cultures from patient samples and compare it with the EUCAST disc diffusion method. For this study, 334 bacterial strains from urinary cultures were used; E. coli (n = 211) E. faecalis (n = 45), Klebsiella spp (n = 37), P. mirabilis (n = 17) and other gram-negative rods (n = 24). The direct resistance method and the EUCAST disk diffusion method were performed in parallel on all bacterial strains and six different types of antibiotics were tested. The diameter of the inhibition zones was measured and categorized according to S, I, R for each antibiotic. From a total of 1674 tested antibiotics 23 discrepancies were found where E. coli represented 13 of these. Mecillinam was the antibiotic where most discrepancies were noted, a total of ten. The agreement between the two methods was 98,6% from 1674 tested antibiotics. The direct resistance method allows a response to the patient 24 hours earlier than the EUCAST disc diffusion method.