Antibiotic resistance (AR), or the ability of bacteria to evade the effects of antibiotics, is a global public health crisis that limits the effectiveness of antibiotics in treating infections. AR is governed by mechanisms in bacteria that serve competitive and ecological purposes. These mechanisms are encoded by genes that can be transmitted from resistant to susceptible bacteria within a population via horizontal gene transfer (HGT) while in the presence of selective pressure. While AR is ancient and ubiquitous, anthropogenic use of antibiotics in medicine and livestock drive the proliferation of AR in bacteria.
Due to the interconnected complex nature of AR spread between human, animals and environment domains, AR should be studied from One Health perspective. Historically, AR has been studied from a clinical and agricultural aspect, but the role of the environment as a reservoir of clinically relevant AR genes and bacteria is under-studied. It has been postulated that the environment plays a critical role in the spread and maintenance of AR, due to the large amount of antibiotics, AR genes, and AR bacteria that enter the environment via human and animal feces. Therefore, it is necessary to characterize AR in the environment and the potential implications for human and animal health.
Chapter 1 is a literature review that summarizes the issue of AR from an environmental and microbial ecology perspective. The scope of the AR crisis from a public health perspective is described, however this review focuses on the role of anthropogenic pollution in promoting the dissemination of AR genes in bacteria, focusing on animal- and human-originated contamination. Differences in the resistome, or the collection of AR genes in a community, between animals, humans, and the environment are described. Finally, due to the interconnected nature between the microbiome and resistome, this review discusses the principles of microbial community ecology in relating those two components. The major knowledge gaps in studies of environmental AR are identified, particularly highlighting the importance of the environmental resistome in clinical AR.
To better characterize the relationship between environmental AR and public health, clinically relevant, carbapenem AR genes and microbial communities are characterized from a surface water ecosystem. Chapter 2 compares the microbial communities of the matrices (compartments) in a multiuse river watershed and identifies those that accumulate carbapenem AR genes to determine the matrices of most concern for carbapenem resistance gene pollution, ecosystem disruption, and potential impacts to human and animal health. DropletDigitalTM PCR is used to quantify three carbapenemase-encoding AR genes (blaKPC, blaNDM, and blaOXA-48). These genes were selected because of their significance in clinical AR infections and gene mobility, as they are plasmid mediated. These genes encode beta lactamase enzymes (bla), specifically Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), and oxacillinase-48-type carbapenemase (OXA-48). Microbial community analysis is completed on a subset of samples with 16S rRNA gene sequencing. Overall, the fish gut and periphyton are highlighted as reservoirs of concern for AR because they accumulate carbapenem resistance genes, host diverse microbial communities, and have natural functions that promote AR transmission and maintenance in the environment.
Because Chapter 2 highlights the importance of periphyton and the fish gut as AR reservoir, Chapter 3 focuses on the characterization of the entire resistome and the linking of bacterial hosts with AR genes in these sample types using metagenomic sequencing. Analyzing the microbiome and resistome together are important to describe the health hazard and clinical relevance of AR from environmental resistomes, as we can identify potentially pathogenic AR bacteria. Microbiomes and resistomes are characterized from periphyton samples via long read sequencing and from fish gut samples via shotgun sequencing. Hosts of AR genes and mobile genetic elements (MGEs) are annotated from the periphyton samples. This study reveals that while the periphyton samples do not contain clinically relevant AR bacteria, they are potential hot spots of gene mobility for environmental microbes, as highlighted by the abundant and diverse MGE community. Furthermore, as food sources for fish and other aquatic organisms (Coles et al., 2012), periphyton are a concern for spreading AR genes and transmissible elements through the aquatic food web. The fish gut is confirmed to not only be a reservoir of carbapenem resistance, but beta-lactam resistant in general, which is of great relevance to public health.
Chapter 4 investigates the effect of wastewater discharge on freshwater ecosystems on a state scale, as WWTPs are a major source of AR to the environment. This study incorporates the methods of chapters 2 and 3 in a different context, as it focuses on wastewater and near-WWTP environments. The goals of this study are to characterize the change in microbiomes and resistomes from wastewater influent to the receiving freshwater, as well as to identify the impacts of wastewater effluent discharge on the surrounding freshwater ecosystem. Therefore, wastewater influent and effluent are analyzed from WWTPs in Cleveland, Columbus, and Cincinnati, OH. These cities are included because they are major urban areas in OH. Surface water, periphyton, sediment, and fish gut samples are analyzed from the freshwater environments near the WWTP outfall. Carbapenem resistance genes (blaKPC, blaNDM, blaOXA-48) and the MGE, IntI1, are quantified via DropletDigitalTM PCR in all samples. IntI1 is quantified as a marker of genetic mobility (McConnell et al., 2018) and anthropogenic pollution (Gillings et al., 2014). A subset of influent, effluent, water, and fish gut samples are selected for shotgun metagenomic sequencing to characterize their microbiomes and resistomes. The most clinically relevant AR genes hosted in pathogens are annotated from wastewater influent. However, pathogenic AR bacteria are still identified from effluent discharge and receiving surface waters, indicating that while WWTPs reduce the clinically relevant AR burden in wastewater, they do not eliminate the public health hazard.
Finally, in Chapter 5, the human gut resistome and microbiome are explored in the context of the household environment. Specifically, this study analyzes the microbiomes and resistomes (via long read sequencing) of infants and young children from rural Nicaragua in the context of household environmental contamination and anthropological factors. While the most significant factors in the developing microbiome and resistome are identified as age and body mass index (BMI), there is a relationship between the quality of the household environment and the developing gut microbiome. Specifically, gut microbiome diversity is greater in children from houses with high dog and ruminant fecal loading of their household floors, determined with microbial source tracking. It is found that Enterobacteriaceae (Escherichia coli) are a major host of AR genes in the developing human gut.
In conclusion, this dissertation presents several studies that describe the relationship between microbiomes, resistomes, and fecal contamination from a One Health perspective, bridging the knowledge gap between clinical and environmental AR. Future studies should continue to characterize potential sources of environmental AR, such as stormwater, in a variety of settings, including rural and low- and middle-income settings. Combatting AR requires a holistic, inclusive, and global perspective, as is encompassed in this dissertation.