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Listeria monocytogenes is food-borne pathogen that causes listeriosis in individuals with a compromised immune system and pregnant women. This pathogen can survive in anaerobic conditions present in specially packaged foods as well as the gastrointestinal tract. The purpose of this study is to evaluate virulence of L. monocytogenes F2365 in anaerobic conditions. Another goal of this study is to establish gerbils as the ideal animal model since descrepancies exist in current models. Gerbils were orally infected with one of four doses: 1) phosphate buffered saline, 2) 5X106CFU aerobic dose, 3) 5X108CFU aerobic dose, and 4) 5X106CFU anaerobic dose. Results indicate anaerobically cultured F2365 colonized the intestines consistently throughout the study unlike aerobic cultures. Additionally, intestinal damage was observed in challenged gerbils. Further goals include evaluating how viurlence is influenced in anaerobic conditions with varying bile concentrations and pH levels.

Microbial community functional diversity enhances the degradation of organic matter and pollutants in the environment, but there is a growing concern that these ecosystem services may be altered by the introduction of emerging environmental contaminants including silver nanoparticles (AgNPs) into aquatic systems. We added 0, 25, 50, 75, 100, and 125 mg L−1 (nominal concentrations) of citrate-AgNP and polyvinylpyrrolidone-AgNP (PVP-AgNP) each to freshwater sediment and examined their antimicrobial effects on microbial communities using community-level physiological profiling. The results showed that citrate-AgNP decreased the overall microbial catabolic activity by 80% from 1.16 ± 0.02 to 0.23 ± 08 while PVP-AgNP decreased the catabolic activity by 51% from 1.25 ± 0.07 to 0.61 ± 0.19 at 125 mg L−1. Citrate-AgNP and PVP-AgNP caused a statistically significant reduction in substrate richness and substrate diversity that decreased microbial functional diversity. AgNPs decreased microbial catabolic capability and functional diversity at concentrations ranging from 0.12 ± 0.04 to 0.43 ± 0.07 mg Ag kg-1 which are lower than the predicted concentrations in freshwater sediment. To our knowledge, this is the first study to demonstrate inhibition of microbial functional diversity by citrate-AgNP and PVP-AgNP in a pathogen impaired stream. Citrate-AgNP caused greater inhibition of carbon substrate utilization but amino acids, carbohydrates, and carboxylic acids were the most affected carbon groups which led to a shift in the metabolic fingerprint pattern of the microbial community. AgNPs decreased the catabolic capability and the ability of the microbial community to degrade organic matter and a variety of pollutants in the environment.

Silver nanoparticles (AgNPs) are tiny particles of silver with nanoscale dimensions (between 1 and 100 nm) and unique antimicrobial properties. AgNPs are potential environmental contaminants increasingly applied in consumer products. The effects on nontarget biological systems are not clearly defined. Research has shown that AgNPs may inhibit the function of bacteria responsible for organic matter decomposition, nutrient cycling, and control of pathogens population in the aquatic system. AgNPs have recently been detected in a treated municipal wastewater raising concerns about their potential risk to aquatic organisms. The microbial community in the sediment has a greater risk of AgNPs exposure, as metals in aquatic systems settle in the sediment. Studies have shown that microbial community growth and carbon sources utilization patterns were altered in response to AgNPs exposure in marine estuarine sediments. The antimicrobial activity of AgNPs in freshwater sediments may be different due to the water chemistry. Few studies have evaluated the toxicity of AgNPs in freshwater sediments due to the complex nature of their water chemistry. The current study investigated microbial community’s responses to AgNPs in sediments collected from a local stream. Microbial growth and activity assays were performed to determine whether AgNPs pose a risk to the microbial community in freshwater sediments. We found that AgNPs inhibited microbial growth, enzyme activity, and catabolic capabilities (P < 0.05). The number of viable bacterial cells and the ability of the microbial community to utilize different carbon sources decreased at 0.431 and 0.538 mg AgNPs kg-1 sediment, which are found within the estimated AgNPs concentration range in sediments. AgNPs inhibited the activity of glucosidase, an enzyme responsible for carbohydrate metabolism, but the activity of alkaline phosphatase was not affected. The current study demonstrates that AgNPs can inhibit the growth and functional diversity of beneficial microorganisms, which may affect the quality of surface waters and their designated uses. These adverse effects are expected due to the demonstrated antimicrobial properties of AgNPs incorporated in several commercial products. Toxicological data generated from this study could be incorporated in ecological risk assessment by regulatory agencies to assess the impacts of AgNPs on ecosystem systems.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22485号 / 農博第2389号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5265(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 吉田 天士, 教授 澤山 茂樹, 教授 菅原 達也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The rapid spread of antibiotic resistance is a major global health concern, jeopardizing the successful treatment of bacterial infections. Natural environments are potential hotspots for the emergence and spread of antibiotic resistance genes (ARGs). Among these potential hotspots, aquatic ecosystems are of particular concern, as they receive wastewater containing antibiotic-resistant bacteria and ARGs originating from both human and animal sources. Several key questions remain to be addressed. What is the fate of ARGs in receiving water bodies? What are implications of environmental ARGs for human health? How does wastewater discharge impacts aquatic microbial communities with regard to the overall ecosystem well-being? The objective of this work was to investigate the impact of wastewater, seasonal variations, and the riverine compartment on the prevalence of selected ARGs and the composition of natural microbial communities in a near-pristine river, and to specifically assess the effect of antibiotics on riverine microbial communities. Quantitative real-time PCR was used to monitor the abundance of three indicator ARGs (sul1 and sul2, conferring resistance against sulfonamide antibiotics, and intI1, a marker for anthropogenic pollution) upstream and downstream from a wastewater treatment plant (WWTP). Furthermore, the impact of WWTP effluent on the riverine microbial community was examined through 16S rRNA amplicon sequencing. Wastewater was the main source of all three target genes and significantly altered the microbial community in the river. The surface water compartment served as a dissemination route for ARGs, with increased prevalence even 13 km downstream of the WWTP, particularly during the summer season when the proportion of wastewater in the river was high. Notably, riverbed biofilms served as a local reservoir for ARGs only at the discharge point, with little abundance of target genes further downstream. The sulfonamide antibiotic sulfamethoxazole (SMX) was persistent in both near-pristine and wastewater-impacted river water when introduced at a concentration of 12.5 µg/L, but had neglectable effects on the microbial community diversity. Interestingly, concentrations as high as 100 µg/L SMX induced a short-term increase in microbial activity in both surface water and biofilm compartment, as revealed by bulk and nanoscale measurements. Altogether, this work underscores the fundamental role of wastewater treatment in combating the environmental dissemination of antibiotic resistance.:Summary 1 Zusammenfassung 5 1 Introduction 9 1.1 Rundown of the global antibiotic resistance crisis 9 1.1.1 History of antibiotics 9 1.1.2 Emergence of antibiotic resistance 9 1.1.3 Integrons as vehicles for antibiotic resistance 10 1.1.4 Risks related to environmental antibiotic resistance 12 1.2 Fate of antibiotic resistance genes in the aquatic environment 14 1.2.1 Genetic indicators for antibiotic resistance 14 1.2.2 River surface water compartment as dissemination route for antibiotic resistance 15 1.2.3 River biofilm compartment as reservoir for antibiotic resistance 17 1.3 Impact of antibiotics on aquatic microbial communities 18 1.4 Fate and effect of sulfamethoxazole in surface waters 20 2 Scope of the thesis 22 3 Main findings and scientific implications 24 3.1 Fate of antibiotic resistance genes after wastewater discharge into a near-pristine river 24 3.1.1 Wastewater is the primary source for aquatic antibiotic resistance 24 3.1.2 Drought increases the antibiotic resistance prevalence in surface waters 25 3.1.3 Riverbed biofilms serve as local reservoirs for antibiotic resistance genes 26 3.2 Anthropogenic pollution is the key driver for microbial community alteration 26 3.3 Sulfamethoxazole increases the microbial activity of aquatic microbial communities 27 4 Conclusions and future perspective 29 5 References 31 6 Publications 43 6.1 Publication 1 43 6.2 Publication 2 56 6.3 Publication 3 69   7 Appendix 94 7.1 Declaration of independent work 94 7.2 List of publications and conference contributions 95 7.2.1 Publications 95 7.2.2 Conference contributions 96 7.3 Contribution of Co-authors 97 7.4 Curriculum vitae 101 7.5 Acknowledgements 104 7.6 Supplementary Material 105 7.6.1 Supplementary Material for Publication 1 105 7.6.2 Supplementary Material for Publication 2 118 7.6.3 Supplementary Material for Publication 3 125

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Bottled drinking water is a common form of water consumption that has grown in popularity and dependency. With countless types and brands available, there are factors to consider regarding the variations in mineral content and microbiological quality, and environmental consequences associated with importing natural bottled waters. Saudi Arabia is the largest desalinated water producer, and although there are scarce natural water resources, consuming locally produced water can alleviate environmental pressures, so long as local bottled water is of good quality and provides the basic function of drinking water. The objective of this study is to scrutinize the variations in bottled waters available in the Saudi market and compare local and imported waters regarding water quality and compliance with health regulations. Surveying local bottled waters revealed that over 60% were imported, adding to the environmental significance. Results from inductively coupled plasma-atomic emission spectroscopy, ion chromatography, and pH meters indicate most bottled waters were compliant with health standards. Flow Cytometry (FCM) was used to determine microbial cell densities. Purified waters, on average, contained significantly lower concentrations than other water types, while mineral waters contained the most cells per milliliter. Microbiological stability was examined through incubating samples at 30°C to promote microbial growth, and results were analyzed using an online FCM system. Purified waters had the least amount of growth and to a lesser degree than mineral and sparkling, which varied in microbial growth responses. DNA extractions and microbial community analyses were performed on select mineral waters before and after incubation. Results showed microbial genera found were similar between samples from the same country and some were unique. All samples exhibited some change in microbial community composition after incubation, signifying that certain communities thrived more than others. Proteobacteria was the most common abundant phylum among samples. Although analysis was restricted to the genera level, there were no health concern associated with types of bacteria discovered. In conclusion, local purified waters had greater microbiological quality and stability than imported waters, and mineral compositions were within the healthy range. Reducing reliance on foreign natural waters may help alleviate environmental consequences and improve local economy

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