This dissertation documents the development of an environmental framework for monitoring antimicrobial resistance gene (ARG) dissemination in the aquatic environment. The work opens with a review of the relevant literature and outlines the importance of an environmental framework for monitoring ARG dissemination as part of antimicrobial resistance risk assessments. The ability to interrogate sequencing data quickly and easily for the presence of ARGs is crucial in order to facilitate their monitoring in the environment. As current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria in the environment were limited in their effectiveness and scope, the dissertation begins by describing the design and implementation of a Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally-acquired ARGs in raw sequencing data. The suitability of metagenomic methods for monitoring the ARG content of effluents from faecal sources was then assessed via a pilot study of a river catchment. Novel metagenomes generated from effluents entering the catchment were interrogated for ARGs. The relative abundance of ARGs in effluents were determined to be higher relative to the background environment, as were sequences relating to human and animal pathogens and mobile genetic elements. Thus, effluents were implicated in the dissemination of ARGs throughout the aquatic environment. To determine if ARGs were potentially in use in the environment, the expression of ARGs within effluents was then evaluated across a series of longitudinal samples through the use of metatranscriptomics, and the presence of potential environmental antimicrobial selection pressures was examined. This demonstrated that the abundance of ARGs, as well as antimicrobial usage at the effluent source, was correlated with the transcription of ARGs in aquatic environments. The work described in this dissertation has also found that horizontally transmitted ARGs were present in pathogenic endospore-forming bacteria commonly found across the aquatic environment, potentially providing a mechanism for ARG persistence in the environment. Finally, these findings were integrated into a universal framework for monitoring ARG dissemination in aquatic environments and used to highlight the developments required to incorporate this framework into future environmental ARG research and to facilitate antimicrobial resistance risk assessments.

Antibiotics are today one of the most important cornerstones in modern healthcare when it comes to treating bacterial infections. It is an asset human kind have been leaning on for the last century, but excessive and widespread misuse of antibiotics have left deep scars in the form of multi resistant pathogenic strains of bacteria that we soon will not be able to treat. A lot of research have been invested in understanding the mechanisms and spread of resistance within bacteria living in planktonic form, overlooking the fact that there are more lifestyles that causes problems. In this study, focus has been put on antibiotic resistance within bacteria living as biofilms, a lifestyle that causes problems in chronic infections and prosthetics/medical implants. By constructing resistant mutants derived from a biofilm forming strain of Escherichia coli, the minimal selection concentration has been investigated in both planktonic and biofilm assays for Streptomycin and Ciprofloxacin. By comparing the results, it is possible to evaluate if and how the antibiotic resistance properties differ between the two lifestyles. Focus has been put on concentrations of antibiotics below the minimal inhibitory concentration with the objective to see how selection of antibiotic resistant mutants take place with the susceptible strain still growing, although with reduced growth rate. The hope is that the results gained in this study will provide a foundation for future research regarding antibiotic resistance in biofilms, and be part of the solution to the excessive resistance problem before it is too late.

Thesis advisor: Eranthie Weerapana / Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of our society and urgently calls for new strategies for infection diagnosis and antibiotic discovery. The overuse and misuse of broad-spectrum antibiotics has contributed to the antibiotic resistance crisis. Additionally, treatment of infections with broad-spectrum antibiotics can cause disruption to the host gut microbiome. The development of narrow-spectrum antibiotics would be ideal to avoid unnecessary cultivation of antibiotic resistance and damage to the human microbiota. Bacteria present many mechanisms of resistance, including modulating their cell surface with amine functionalities. In an age where infections are no longer responding to typical antibiotic treatments, novel drugs that target the characteristics of antibiotic resistance would be beneficial to remedy these defiant infections. Herein, we describe the utility of iminoboronate formation to target the amine- presenting surface modifications on bacteria, particularly those that display antibiotic resistance. Specifically, multiple 2-acetylphenylboronic acid warheads were incorporated into a peptide scaffold to develop potent peptide probes of bacterial cells. Further, by engineering a phage display library presenting the 2-acetylphenylboronic acid moieties, we were able to perform peptide library screens against live bacterial cells to develop reversible covalent peptide probes of target strains of bacteria. These peptide probes, which were developed for clinical strains of Staphylococcus aureus and Acinetobacter baumannii which display resistance, can label the target bacterium at submicromolar concentrations in a highly specific manner and in complex biological milieu. We further show that the identified peptide probes can be readily converted to bactericidal agents that deliver generic toxins to kill the targeted bacterial strain with high specificity. It is conceivable that this phage display platform is applicable to a wide array of bacterial strains, paving the way to facile diagnosis and development of strain-specific antibiotics. Furthermore, it is intriguing to speculate that even higher potency binding could be accomplished with better designed phage libraries with dynamic covalent warheads. This work is currently underway in our laboratory. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Twenty-eight Holstein and crossbred calves of both genders were used to evaluate the effect of milk replacer antibiotics on abundance of selected antibiotic resistance genes (ARG) in the feces. Calves were blocked by breed, gender, and birth order, and assigned to one of three treatments at birth. Treatments were control (containing no antibiotics in the milk replacer), subtherapeutic (neomycin sulfate and oxytetracycline hydrochloride each fed at 10 mg/calf/d), and therapeutic (no antibiotics in the milk replacer until d 36, then neomycin sulfate and oxytetracycline hydrochloride each fed at 1000 mg/calf/d for 14 d). Calves were fed milk replacer twice daily at 0600 h and 1800 h. Fecal and respiratory scores and rectal temperatures were recorded daily. Calves were weighed at birth and weaning to calculate average daily gain. Beginning at six weeks of age fecal grab samples were collected from heifers at 0600 h, 1400 h, 2000 h, and 2400 h for 7 d, while bull calves were placed in metabolism crates for collection of all feces and urine. DNA was extracted from feces, and ARG corresponding to the tetracyclines (tetC, tetG, tetO, tetW, and tetX), macrolides (ermB, ermF), and sulfonamides (sul1, sul2) classes of antibiotics along with the class I integron gene, intI1, were measured by quantitative polymerase chain reaction (qPCR). No tetC or intI was detected. There was no significant effect of antibiotic treatment on the absolute abundance (gene copies/ g wet manure) of any of the ARG except ermF, which was lower in the antibiotic-treated calf manure probably because host bacterial cells carrying ermF were not resistant to tetracycline or neomycin. All ARG except tetC and intI were detectable in feces from 6 weeks onwards, and tetW and tetG significantly increased with time (P < 0.10), even in control calves. Overall, the majority of ARG analyzed for were present in the feces of the calves regardless of exposure to dietary antibiotic. Feed antibiotics had little effect on the ARG monitored; other methods for reducing the ARG pool should also be investigated. / Master of Science

Thesis advisor: Tim van Opijnen / Each year, the opportunistic pathogen Streptococcus pneumoniae causes millions of illnesses and nearly 300,000 deaths worldwide. Despite widespread vaccination campaigns, S. pneumoniae persists as a public health risk in large part to its high genomic diversity. In previous work, our group has shown that functional pathways, including stress response to antibiotics, are not necessarily conserved between pneumococcal strains. Thus, a holistic pangenome view of S. pneumoniae is a promising avenue to gain understanding of the species and to inform clinical treatment methods. Our group has selected 36 strains, covering 78% of known pneumococcal genetic diversity, for S. pneumoniae pangenome studies. We have previously constructed transposon libraries and performed Tn-seq for 22 of these strains in both in vitro and in vivo conditions. From these studies, our group has constructed pangenome profiles of genes essential for reproduction in culture conditions, infection in a mouse model, and attachment in a human nasopharyngeal epithelial cell line. In this study, we develop and execute a pipeline to construct iSP20, a set of in silico metabolic models for 34 S. pneumoniae strains. We employ these models to predict nutrient and metabolic gene essentiality on both the strain and pangenome level, demonstrating that key patterns in the strains’ essentialomes translate to a metabolic context. Additionally, we perform a functional analysis of the metabolic models, revealing a highly connected metabolic genome and essentialome. We uncover differences in the in vitro and in silico core essentialomes and identify potential sources of discrepancy between the two datasets. Overall, this work demonstrates the utility of strain-specific metabolic models in pangenome essentiality studies and provides enhanced understanding of metabolism in S. pneumoniae. / Thesis (BS) — Boston College, 2020. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

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With the rise of antibiotic resistance, there is ongoing need to find new antibiotics. As bacteria develop resistance to the current classes of antibiotics available, it is imperative to discover new ways to target bacteria. In this thesis, I focused on one of the basic components that all bacteria need to survive: a source of carbon. Here, I explore whether we can exploit this aspect for drug discovery. For bacteria to colonize a host and cause an infection, it must first be able to meet its nutritional needs for growth. Different host infection sites will have different carbon sources available. Some sites, like the gut, will have commensal bacteria which will compete with invading pathogens for carbon sources. While we still lack understanding of the specific growth environment bacteria experience during infection, it is important to understand how bacteria grow when given different nutrients. For the first part of my work, I systematically probed the gene essentiality patterns of E. coli grown in different carbon sources. I generated a large dataset of growth phenotypes that I compiled into a user-friendly web-application, Carbon Phenotype Explorer (CarPE). I identified many poorly annotated genes, and further characterized the gene ydhC as an adenosine transporter. After characterizing how the growth of E. coli and the genes essential for survival change depending on each carbon source, I looked at whether antibiotic efficacy changed depending on the carbon source used. I found that growth in oxaloacetate alters the proton motive force and potentiates macrolide antibiotics. I also found that linezolid, a compound that does not work on gram-negative bacteria due to efflux, is more effective when adenosine is the carbon source. Together, this work forms a foundation for future research into studying how carbon sources can be exploited in the field of antibiotic discovery. / Thesis / Candidate in Philosophy / There is an urgent need for new antibiotics. Previous antibiotic discovery has primarily been conducted on bacteria growing in nutrient rich laboratory conditions. This led to antibiotics that targeted the same few bacterial processes. However, since bacteria need to survive in a host to cause an infection, there are targets that may be viable during an infection that we miss by using standard laboratory media. Bacteria need a source of carbon to survive, and each infection site contains different chemicals that bacteria can use as a source of carbon. My work studies how bacteria grow in the presence of different carbon sources. First, I systematically tested which bacterial genes are required for E. coli to grow in 30 different carbon sources. I then examined the effectiveness of antibiotics on bacteria grown using these different carbon sources. Together, this work helps us understand how changing carbon sources in the growth media we use to cultivate bacteria can change which genes are required and how it may change how bacteria survive antibiotic stress. When we discover the specific compositions of host infection environments, we can leverage this knowledge to find antibiotics that target these carbon acquisition pathways in bacteria.

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While antibiotics have saved the lives of millions of people since the discovery of the first β-lactam, penicillin, their continued effectiveness is being increasingly threatened by resistant bacteria. Bacterial resistance to β-lactams is mainly achieved through the production of serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs). Although both types of β-lactamases are commonly isolated in clinical settings, MBLs represent the greatest threat to public health since they are resistant to SBL inhibitors and most β-lactams. However, aspergillomarasmine A (AMA), a fungal natural product synthesized by Aspergillus versicolor, was shown to be a rapid and potent inhibitor against two clinically relevant MBLs: NDM-1 and VIM-2. In bacteria possessing these enzymes, AMA could rescue the activity of meropenem, a broad-spectrum β-lactam that is usually reserved for the treatment of the most severe bacterial infections. However, many questions remain revolving around AMA's inhibitory potency and spectrum. Therefore, the activity of AMA in combination with six β-lactams from three subclasses (carbapenem, penam, cephem) was explored against 19 MBLs from three subclasses (B1, B2, B3). After determining that AMA activity was linked to MBL zinc affinity and that AMA was more potent when paired with a carbapenem, the efficacy of an AMA/meropenem combination was evaluated with and without avibactam, a potent SBL inhibitor. This study used ten Escherichia coli and ten Klebsiella pneumoniae laboratory strains as well as 30 clinical strains producing at least one MBL and one SBL. Once establishing that the AMA/avibactam/meropenem combination was effective against carbapenemase-producing Enterobacterales, new Acinetobacter and Pseudomonas shuttle vectors were created. With these shuttle vectors, it was determined that the AMA/avibactam/meropenem combination was effective against some of the bacteria topping the World Health Organization’s priority pathogen list. / Thesis / Doctor of Philosophy (PhD) / Bacteria are all around us. While some bacteria can promote human health, others can cause serious infections. These infections are typically treated with antibiotics. β-Lactam antibiotics, such as penicillins and cephalosporins, are especially important to medicine. Unfortunately, an increasing number of bacteria employ enzymes, known as β-lactamases, which negate the effects of β-lactam antibiotics. Previous studies demonstrated that a natural product, known as aspergillomarasmine A (AMA), could inhibit some β-lactamase enzymes. Consequently, the inhibitory power of AMA was further explored against a larger number of β-lactamase enzymes and in combination with different β-lactam antibiotics. After discovering that AMA had more inhibitory power when combined with a β-lactam antibiotic known as meropenem, the efficacy of the AMA/meropenem pairing was evaluated against resistant bacteria in the presence and absence of avibactam, another β-lactamase inhibitor. The AMA/avibactam/meropenem combination was shown to be effective against some of the world’s most antibiotic-resistant bacteria.