Bacterial resistance to antibiotics has become a worldwide health problem, resulting in untreatable infections and escalating healthcare costs. Wastewater treatment plants are a critical point of control between anthropogenic sources of pathogens, antibiotic resistant bacteria (ARBs), antibiotic resistance genes (ARGs), and the environment through discharge of treated effluent and land application of biosolids. Recent studies observing an apparent resuscitation of pathogens and pathogen indicators and the widening realization of the importance of addressing environmental reservoirs of ARGs all lead toward the need for improved understanding of ARG fate and pathogen inactivation kinetics and mechanisms in sludge stabilization technologies. This research has investigated the fate of two pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, and various ARGs under pasteurization, anaerobic digestion, biosolids storage, and land application conditions. Pathogen die-off occurs at a rate specific to each pathogen and matrix in ambient and mesophilic temperature environments. Viable but nonculturable (VBNC) states are initiated by thermal treatments, such as thermophilic digestion and possibly pasteurization, and allow the persistence of pathogen cells and any ARGs contained therein through treatment and into the receiving environment where resuscitation or transformation could occur. Raw sludge ARG content does affect digester effluent quality, although the predominant mechanisms of ARG persistence may be different in mesophilic versus thermophilic digestion. In both thermophilic and mesophilic digestion, a correlation was observed between raw sludge and digester ARGs associated with Class 1 integrons, possibly as a result of horizontal gene transfer. ARB survival was shown to contribute to ARG content in mesophilic digestion, but not thermophilic digestion. Thermophilic digestion may achieve a higher ARG reduction because of reduced microbial diversity compared to mesophilic digestion. However, it is evident that horizontal gene transfer still does occur, particularly with highly mobile integrons, so that complete reduction of all ARGs would not be possible with thermophilic digestion alone. Surprisingly, the experiments that introduced various concentrations of antibiotic sulfamethoxazole and antimicrobial nanosilver did not induce enhanced rates of horizontal gene transfer. Finally, ARG concentrations in biosolids increased during cold temperature storage suggesting that there is a stress induction of horizontal gene transfer of integron-associated ARGs. / Ph. D.

Food-borne illness outbreaks are occasionally associated with fresh-vegetable consumption, in part due to lack of a microbial inactivation step before consumption. Raw manure or improperly composted manure applied as soil amendments is an established source of pathogenic bacterial contamination. However, less is known about whether such soil amendments could serve as a source of transmission of antibiotic-resistant bacteria (ARB) or antibiotic-resistance genes (ARGs) via fresh produce. As such knowledge is developing, it is useful to identify strategies for mitigating ARGs and ARB on vegetable surfaces, especially those that are synergistic with known benefits in terms of general pathogen reduction on fresh produce. Sanitizers play an important role in post-harvest processing of vegetables, especially in terms of disinfecting the wash water and preventing cross-contamination. Further, temperature and time of storage of vegetables are critical to prevent the growth of microorganisms. To provide a background inoculum representing potential pre-harvest carryover of ARB and ARGs, carrots or romaine lettuce leaves were dipped in a slurry derived from composted manure from dairy cows previously dosed with antibiotics and further inoculated with multi-drug resistant E. coli O157:H7, a human pathogen, and a spoilage-associated and opportunistic pathogenic strain of Pseudomonas aeruginosa. Inoculated carrots (n=3, 25 g) were washed with water containing different sanitizers (sodium hypochlorite or peroxyacetic acid) or unwashed (control), packaged and stored at 10ºC for 7d or 2ºC for up to 60 d. Inoculated lettuce leaves (n=3, 100 g) were washed with sodium hypochlorite, packaged in modified atmosphere conditions (98% nitrogen), irradiated (1.0 kGy) and subsequently stored at 4ºC for 14 d. The effect of post-harvest treatment were compared at various times by enumeration on selective media. In addition, cultureindependent techniques were also performed to determine changes to the surficial carrot and lettuce microbiota by sequencing bacterial 16S rRNA gene amplicons. The effect of post-harvest treatments on the types and relative abundance of ARGs, also known as the “resistome,” were profiled by shotgun metagenomic sequencing and qPCR. Addition of a sanitizer during wash, storage temperature, and duration of storage affected the bacterial community structures on carrots, represented by the weighted Unifrac distance matrices (ANOSIM, R=0.465). Storage of sanitizer-washed carrots at 10ºC was associated with an increase in relative abundance of Pseudomonadaceae compared to 2ºC storage for 7 d (Wilcoxon, p<0.05). Increase in storage temperature from 2ºC (optimum) to 10ºC (temperature abuse) of sanitizer-washed carrots resulted in enrichment of ARGs conferring resistance to the following antibiotic classes: multidrug, peptide, polymyxin, quinolone, triclosan, aminoglycoside, bacitracin, β-lactam, and fosfomycin. Irradiation resulted in significant reductions (~3.5 log CFU/g) of inoculated antibiotic-resistant E. coli O157:H7 and Pseudomonas sp. on lettuce surfaces (ANOVA, p<0.05). The lettuce resistome, represented by the Bray-Curtis similarity of ARG occurrence, was affected by irradiation (ANOSIM, R=0.406). Irradiation of lettuce followed by 14 d of storage at 4ºC resulted in 2-4-fold reductions in relative abundance of ARGs encoding resistance to the following antibiotic classes: triclosan, quinolones, multidrug, polymyxin and β-lactam (Wilcoxon, p<0.05). No additional increase or reduction of the tet(A) gene present on inoculated P. aeruginosa was evident after 14d storage at 4ºC on irradiated samples. Results of this study suggest that inclusion of a sanitizer in wash water, irradiation, and storage at optimum refrigerated temperatures may offer effective strategies to combat proliferation of antibiotic resistant bacteria and antibiotic resistance genes on fresh produce. Further research is needed develop interventions that can mitigate tet(A) and other ARGs on produce that were not significantly reduced by irradiation. This study will guide future research on microbiome and metagenome of processed produce and assessment of critical control points to reduce the risk of antibiotic resistance from farm-to-fork. / PHD / Post-harvest interventions; such as washing, irradiation and cold storage, are employed to provide safe and wholesome fresh vegetables to consumers. Washing of vegetables in water that includes a sanitizing agent, such as chlorine or peroxyacetic acid (POAA), removes soil from the surface, reduces the bacteria in wash water and prevent cross-contamination between vegetables. It has an additional benefit to reduce microorganisms on produce surfaces that may cause the vegetables to spoil or result in illness in humans. Low temperature storage of produce, usually 0-5ºC, decreases the respiration rate of vegetables and reduces growth of microorganisms during storage. Some of the spoilage and/or pathogenic bacteria may also be antibiotic-resistant, which are commonly termed as antibiotic-resistant bacteria (ARB). Antibiotic resistance is a significant public health concern that leads to ineffective medical treatments, prolonged duration of illnesses and increased hospitalization costs. Antibiotic resistance is encoded by genes that confer resistance to wide range of antibiotic classes, including antibiotics used to treat human illnesses. These genes are termed as antibiotic resistance genes (ARGs). In this study we examined the effect of three common post-harvest interventions, washing with sanitizers, gamma irradiation, and cold storage to reduce antibiotic-resistant bacterial pathogens and antibiotic-resistant spoilage bacteria on carrots and lettuce. Storage temperature, inclusion of sanitizer in wash water, and length of chilled storage significantly influenced the diversity of bacteria found on carrot surface. Inclusion of either sanitizer in the wash water significantly reduced the populations of antibiotic-resistant E. coli O157:H7 (a pathogenic bacterium that causes a dangerous form of gastrointestinal illness) and Pseudomonas sp. (a bacterial species that commonly causes food spoilage). Storage at recommended temperature (2ºC) did not allow these bacteria to regrow and also reduced total ARGs on carrot surfaces. Washing of lettuce with sodium hypochlorite followed by irradiation (1.0 kGy) and storage at recommended temperature (4ºC) were effective in reducing the populations of antibiotic-resistant E. coli O157:H7 and Pseudomonas sp., and additionally reduced the number of some ARGs conferring resistance to select classes of antibiotics, including triclosan, quinolones, multidrug, polymyxin and β-lactam antibiotics on the lettuce surface. A novelty of this research is that it employed new, cutting-edge “metagenomic” DNA sequencing technique to identify and track antibiotic resistance through the various post-harvest interventions. Overall results of this research suggest that inclusion of sanitizer in wash water for fresh produce, followed by storage at refrigerated temperatures below 4ºC may reduce the risk posed by antibiotic resistant bacteria and antibiotic resistance genes on produce.

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 / Anaerobic digestion is a biological treatment technology used on farms to treat manure. It can be used to reduce potential environmental damage from contaminants and manure, homogenize manure for fertilizer, and produce methane gas for renewable energy. An emerging challenge in manure management is the presence of antibiotics such as ceftiofur used in animal production to prevent and treat illnesses. When antibiotics are used on livestock, they are excreted from the animal in manure. When the manure is added to the digester, the antibiotic molecules within the manure can kill the bacteria responsible for manure homogenization and gas production. Ceftiofur is a type of cephalosporin antibiotic used to treat dairy cows for mastitis, a bacterial infection of the udder. When the cows are treated with Ceftiofur, it can remain in the excreted manure and enter the digester. The use of antibiotics on farms is also leading to a global phenomenon known as antibiotic resistance. The bacteria that are exposed to antibiotics can develop mutations to become immune to the antibiotic, and can spread the mutations through antibiotic resistant genes (ARGs). ARGs can spread to bacteria which have never been exposed to antibiotics, making them resistant. This causes a significant concern in regards to disease treatment across the world as the efficacy of antibiotics is threatened. Understanding how ARGs move and how they can be eliminated is crucial to preventing global antibiotic resistance. The purpose of this study was to assess the effect of anaerobic digestion on Ceftiofur and ARGs. Four continuous lab-scale anaerobic digesters, two using control manure and two using manure from cows treated with Ceftiofur, were run at 55˚C for a period of 50 days. Over time, the reactor with manure from cows treated with the Ceftiofur antibiotic produced less gas as compared to the control digesters. The amount of Ceftiofur within the digesters decreased over time, demonstrating anaerobic digestion’s ability to degrade the antibiotic molecule. The effect of anaerobic digestion on the ARG concentration was gene specific. The concentration of the tet(Q) gene, a gene responsible for resistance against the very common antibiotic tetracycline, was reduced by anaerobic digestion. These results demonstrate that anaerobic digestion is a technology which can reduce the environmental impact of manure from Ceftiofur-treated cows. This shows that manure treatment can be a first step in combating antibiotic resistance across the globe.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第25269号 / 工博第5228号 / 新制||工||1997(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 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 Agricultural Science / 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.