Wastewater and bioreactors in wastewater treatment plants (WWTPs) are reservoirs for antibiotic resistance genes (ARGs), which can pass through treatment processes and migrate into the environment as they are released in effluent. WWTPs harbor a variety of ARGs that can be spread by horizontal gene transfer facilitated by mobile genetic elements (MGEs). However, little research has investigated how ARG abundances and mobilization trends differ in suspended versus biofilm growth WWTP bioprocesses, or how ARGs are associated with the presence of antibiotic production genes (APGs) in these treatment systems. The overall objectives of this thesis were to investigate ARGs, MGEs, APGs, viruses, and associated bacterial communities in different types of wastewater treatment systems at various scales. Antibiotic resistance is an urgent public health issue that can spread through the environment via ARGs, particularly those that are associated with MGEs. Using shotgun metagenomics sequencing, we analyzed diversity and abundance of ARGs, APGs, and MGEs in two WWTPs, one with a suspended growth activated sludge treatment process and the other with an attached growth trickling filter biofilm process. We found that abundance and persistence of different types of ARGs varied between the two WWTPs. The proportions of ARGs associated with MGEs were significantly greater in effluents compared to influents in both plants. Furthermore, viruses influence microbial communities within aquatic environments such as WWTPs, but while existing research has characterized lytic viruses in wastewater samples collected with the intention of excluding bacterial DNA, prophages and their associations with bacterial hosts have not been well characterized in WWTPs. These associations are important because viruses can help shape microbial community composition and can influence horizontal gene transfer. To address this knowledge gap, metagenomes from the two full-scale suspended growth and biofilm WWTPs that were previously analyzed for ARG and MGE content were further investigated to characterize diversity and community composition of viral sequences, predominantly consisting of prophages, as well as the bacteria associated with these viral sequences. PERMANOVA analysis of viral beta diversity showed significant differences both between WWTPs and between specific sampling locations within the WWTPs. This suggests that the potential for viral communities to shape microbial communities was different for each sampling location. Community structure of total bacteria was also found to be significantly different than community structure of bacteria associated with viral sequences, thus only certain bacterial taxa were likely to be associated with viruses that can influence their chromosomal DNA and potentially be triggered to lyse the cells. This analysis of ARGs and viruses was further extended to a pilot-scale wastewater treatment system in Israel that treats hospital wastewater. Hospital wastewater is expected to contain elevated abundances of ARGs and antibiotic-resistant bacteria, as well as a diverse assortment of viruses that may shape overall microbial community structure, yet the fate of ARGs throughout hospital wastewater treatment systems and potential connections between bacteria that harbor ARGs and bacteria hosting viruses are not well understood. We collected samples from a transect of the pilot-scale system using both bacterial and viral sampling methods and used shotgun metagenomics to characterize ARGs and bacteria containing ARGs, as well as viruses and the bacterial taxa predicted to host them. A diverse assortment of ARGs were identified throughout the pilot-scale system. Overall removal efficiency of ARGs on a per-cell basis was only 16%, which was substantially lower than ARG removal we previously observed in full-scale municipal WWTPs where overall removal was greater than 90% per cell. Community composition of bacteria harboring ARGs, bacteria putatively hosting viruses, and overall bacterial communities were significantly different from one another based on beta diversity. This shows that particular species of bacteria are more likely to harbor and spread ARGs than others. Additionally, no strong links between viruses and ARGs were observed, suggesting that viruses were not significant vectors for the spread of antibiotic resistance in this system. While it is known that wastewater treatment biofilms contain ARGs, the spatial distribution of ARGs and MGEs as a function of biofilm depth has not been previously explored. In order to analyze vertical heterogeneity of ARG and MGE abundances and diversity in wastewater treatment biofilms, we grew biofilms in a lab-scale rotating annular reactor fed synthetic wastewater influent. We cryosectioned the biofilms into layers representing base, middle, and top portions of biofilms, then used qPCR to analyze abundances of three ARGs commonly found in WWTPs including sul1, ermB, and qnrS, as well as the integron-integrase intI1. We also used 16S rRNA gene amplicon sequencing to analyze differences in community diversity over time and with depth in the biofilm. Results showed that sul1 and intI1 were significantly more abundant in upper biofilm layers near the biofilm-fluid interface compared to bottom layers near the substratum, while ermB and qnrS were only detected in relatively low abundances. Community structure and diversity varied over time and by layer. These results have significant implications as upper regions of biofilms nearest to the biofilm-fluid interface detach more easily than deeper regions of biofilms near the substratum, so portions of biofilms near the biofilm-fluid interface are more likely to move downstream and influence genetic composition of water and biomass in later stages of treatment systems, and have an increased chance of being transferred into receiving water bodies via effluent discharge. Overall, the work presented in this thesis shows that ARGs, including some with high clinical relevance, were diverse, abundant, and often mobile in a variety of types of WWTPs that treat both municipal and hospital wastewater. Viruses were also diverse in these systems and were not found to be significantly associated with ARGs. This work is important because it expands understanding of ecology and genomic context of ARGs in urban wastewater infrastructure. It provides data necessary for understanding fate of ARGs and can inform future research on dissemination of ARGs and their potential risks to public health.

Creator

• Petrovich, Morgan

DOI

• https://doi.org/10.21985/n2-db2y-t684

Subject

• Microbiology
• Environmental engineering
• Bioinformatics

Language

• en

Alternate Identifier

• etdadmin_upload_671878
• http://dissertations.umi.com/northwestern:14727

Keyword

• viruses
• antibiotic resistance genes
• biofilms
• wastewater
• mobile genetic elements

Date created

• 2019-01-01

Resource type

• Dissertation

Rights statement

• In Copyright