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<p> </p> <p>Disinfectant application on environmental surfaces is generally accepted to be effective</p> <p>against vegetative pathogens. However, several important application considerations are not being evaluated</p> <p>in the registration of these disinfectant solutions. This thesis looks into factors impacting the disinfectant wipes' bactericidal and fungicidal efficacy on hard, non-porous surfaces and how the interaction between the disinfectant solution and wipe substrate lead to changes in the efficacy of disinfection.</p>

<p dir="ltr">Human babesiosis is a malaria-like, tick-borne infectious disease of major public health importance with a global distribution. Babesiosis is caused by intraerythrocytic, apicomplexan parasites of the genus Babesia. In the United States, human babesiosis is primarily caused by Babesia microti and Babesia duncani. Of these parasites, B. duncani infection is lethal to susceptible patients. Current treatment for babesiosis includes either the synergistic use of atovaquone and azithromycin or the combination of clindamycin and quinine. However, the side effects and the resistance posed by these parasites called for alternative approaches for the treatment of human babesiosis. Parasite-derived proteases play several functions in the context of parasitic lifestyle and regulate basic biological processes including cell death, cell progression and cell migration. We hypothesized that proteases are promising class of drug targets in Babesia parasites. Using the SYBR-Green assay, we screened a protease inhibitor library consists of 160 compounds against B. duncani in vitro culture at 50µM and identified 13 preliminary hits. Additionally, dose response assays of hit compounds against <i>B. duncani</i> and <i>B. microti</i> in vitro cultures identified 5 compounds as effective inhibitors against parasite growth. Of these 5 compounds, we chose ixazomib, a proteasome inhibitor as a potential drug for further studies based on its lower IC50 of 58nM as well as a higher therapeutic index as compared to other hit compounds. We demonstrated that in a mouse model infected with <i>target,</i>, the most effective inhibitor, the prodrug of ixazomib at a low dose of 2.5mg/kg lowers parasite proliferation without causing any adverse effects in animals. Thus, our studies suggest that Babesia proteasome may be an important drug target, and ixazomib may be a potential compound that may be used for the treatment of human babesiosis.</p>

<p dir="ltr"><i>Serratia marcescens </i>is a bacterium with a ubiquitous environmental distribution and the ability to cause opportunistic infections. This research explores three different group behaviors in <i>S. marcescen</i>s. These are biofilm formation, microbial hitchhiking, and responses to cannabinoid-induced stress. To study biofilm development, we used a crystal violet assay to measure biofilm and compared that to the bacterial growth of those cultures. We looked at the role of nutrients and temperature in biofilm produced by <i>S. marcescens</i> and tested four <i>S. marcescens</i> strains. We found that there were differences in the ratio of biofilm to growth when <i>S. marcescens</i> was grown in different media. The exact relationship between temperature and media composition requires more information than could be attained from these studies. Next, we wanted to investigate whether <i>S. marcescens</i> could also utilize movement of other, more highly motile species of bacteria through a process called microbial hitchhiking. <i>S. marcescens</i> was grown with a highly motile <i>Paenibacillus</i> sp. isolate. <i>S. marcescens</i> was tracked by the red pigment that it produces. It was observed that <i>S. marcescens</i> consistently spread farther across a surface when it was co-cultured with <i>Paenibacillus</i> sp. than when grown alone. This was repeated with three other <i>S. marcescens</i> strains and three different species of <i>Paenibacillus.</i><i> </i>Hitchhiking behavior was observed in all cases. To understand the mechanism by which <i>S. marcescens</i> hitchhikes on <i>Paenibacillus </i>spp., a <i>S. marcescens </i>flagellar mutant was used. Even in the absence of a flagellum, <i>S. marcescens</i> was still able to hitchhike implying that another mechanism must be involved. Finally, we evaluated the response of <i>S. marcescens </i>to cannabidiol (CBD) a phytocannabinoid with antimicrobial and antibiofilm properties, though it has limited potency against Gram-negative bacteria like <i>S. marcescens</i>. We found that CBD did not kill <i>S. marcescens </i>nor did it affect its biofilm development. Interestingly, <i>S. marcescens </i>cultures showed enhanced pigment production in response to high-dose CBD exposure compared to vehicle controls. This response was also observed with exposure to three additional phytocannabinoids. Results from these studies add to our understanding of how <i>S. marcescens</i> can access new areas and persist in a broad range of environments.</p>

<p dir="ltr">The Centers for Disease Control and Prevention’s 2019 Antibiotic Resistance Threats Report highlights more than 2.8 million antibiotic infections each year, with at least 35,000 deaths per annum attributed to antibiotic resistance. The CDC’s 2022 COVID-19 Impact Report emphasizes a 15% increase in hospital-acquired resistant infections between 2019 and 2020, many which are caused by Gram-negative bacteria, bacteria characterized by two encapsulating membranes. The limited treatment options for Gram-negative bacterial infections underscore the critical need for new strategies to combat these pathogens. The β-barrel assembly machinery complex (BAM) is a protein complex located in the outer membrane (OM) of Gram-negative bacteria, facilitating the folding and insertion of β-barrel outer membrane proteins (OMPs) into the OM. Inhibiting the function of this complex is lethal for Gram-negative bacteria, making BAM a significant and promising drug target.</p><p dir="ltr"><i>Fusobacterium nucleatum</i> is a Gram-negative pathogen that functions in the oral microbiome, interacting with multiple levels of biofilm colonizers. <i>F. nucleatum</i> causes oral infections and is linked to colorectal cancer, impacting treatment response and disease recurrence. The pathogenicity of <i>F. nucleatum</i> in both biofilm formation and in cancer involves OMPs whose biogenesis relies on BAM; however, BAM has not been characterized in this organism. The goal of our study here is to better understand the composition, structure, and function of BAM and its potential as a drug target for <i>F. nucleatum</i>. We first used bioinformatics analysis and proteomics to investigate the putative composition of the BAM complex in <i>F. nucleatum</i>. While the core component BamA was identified, there was a notable absence of other typical accessory proteins in this organism's genome. Therefore, we postulate that unlike other bacteria such as <i>E. coli</i> and <i>A. baumannii</i>, the biogenesis of OMPs in <i>F. nucleatum</i> is mediated solely by BamA without the need of accessory components.</p><p dir="ltr">To investigate how BamA can accomplish OMP biogenesis itself, we employed biophysical techniques to analyze the structure of <i>Fn</i>BamA. We resolved the cryo-EM structure of <i>Fn</i>BamA in complex with several Fabs which showed novel structural features not previously observed in bacteria. In these structures, <i>Fn</i>BamA was found to contain four N-terminal POTRA domains arranged in a J-shaped conformation, rather than elongated. The Fab was found to bind primarily along POTRA 3 which likely stabilizes the unique conformation of the POTRA domains. The C-terminal 16-stranded b-barrel domain was observed as an inverted dimer, with the dimer interface mediated by direct interaction of the b1 strands along the lateral seam of both barrel domains. Additionally, we determined the X-ray crystal structure of the barrel domain alone which was found as a monomer. Measurements of the barrel domain of <i>Fn</i>BamA reveal it has a different shape and size than is found in other BamA structures such as in <i>E. coli</i>. Together, these structural differences provide clues to how <i>Fn</i>BamA alone may accomplish OMP biogenesis when additional components are required in other bacteria. Our ongoing studies aim to further characterize the molecular structure and function of <i>Fn</i>BamA in conjunction with promising antibiotics and other putative BAM components if discovered.</p>

The maternal liver exhibits robust adaptations to pregnancy to accommodate the metabolic needs of developing and growing placenta and fetus by largely unknown mechanisms. We found that achaete-scute homolog 1 (Ascl1), a basic helix-loop-helix transcription factor essential for neuronal development, is highly activated in maternal hepatocytes during the second half of gestation in mice. Our aim is to investigate whether and how Ascl1 plays a pregnancy-dependent role. We deleted the Ascl1 gene in the maternal liver using three independent mouse models from mid-gestation until term and identified multiple Ascl1-dependent phenotypes. When Ascl1 was deficient in maternal hepatocytes, maternal livers exhibited aberrant hepatocyte histology, fat accumulation, increased hepatocyte cell cycle, and enlarged size, accompanied by reduced albumin production and elevated levels of free fatty acids, ALT, and AST in the maternal blood, indicating maternal liver dysfunction. In the same situation, maternal spleen and pancreas displayed marked enlargement without an overt structural change; the placenta exhibited striking overgrowth with increased ALP production; and the cecal microbiome showed alterations in the relative abundance of several bacterial subpopulations. Moreover, litters born from maternal hepatic Ascl1 null mutated dam experienced abnormal postnatal growth after weaning. RNA-seq analysis revealed Ascl1-regulated genes in the maternal liver associated with Ascl1-dependent phenotypes. Of particular interest, we found that, in maternal hepatocytes, Ascl1 loss-of-function caused the activation of paternally imprinted gene insulin-like growth factor 2 (Igf2) encoding a major placental and fetal growth factor. IGF2 is also a known mitogen for hepatocytes and several hematopoietic lineages. Thus, IGF2 is a potential inducer of Ascl1-dependent phenotypes including placental overgrowth and maternal organ enlargement. Our studies revealed Ascl1 as a novel regulator of maternal liver physiology during pregnancy. Ascl1 activation in maternal hepatocytes is essential for normal placental growth and appropriate maternal organ adaptations, ensuring the health of both the mother and the fetus.<br>

<p>In the past few decades, invasive mycosis has become a growing threat to global health, afflicting millions of people and claiming the lives of more than 1.5 million patients every year. Moreover, the economic burden of mycotic infections has become increasingly exhausting especially with the recent increases in the number of the high-risk population, the immunocompromised individuals. In the USA, the cost incurred by mycotic infections was estimated to be of more than $7.2 billion only in 2017. Of particular concern, <i>Candida</i> species are the most common fungal pathogens that infect humans, resulting in considerable morbidities and mortality rates that often exceed 50%. Unfortunately, the antifungal drug discovery is currently unable to keep pace with the urgent demand for more effective therapeutic options. Further complicating the situation is the recent emergence of multidrug-resistant species such as <i>Candida</i> <i>auris</i>, triggering outbreaks of deadly Candidemia across the globe. Given the risks inherent to the traditional de-novo drug discovery, combinatorial therapeutics stands out as a promising tool to hamper drug resistance and extend the clinical utility of the existing drugs. In this study, we assembled and screened ~3147 FDA-approved drugs and clinical molecules against fluconazole-resistant <i>C. albicans</i> and <i>C. auris</i> isolates, for the aim of restoring the antifungal activity of azole antifungals against drug-resistant <i>Candida </i>species. The screen revealed five promising hits: pitavastatin (antihyperlipidemic), ospemifene (estrogen receptor modulator), sulfa antibacterial drugs, lopinavir (antiviral), and aprepitant (antiemetic).</p> <p>All identified hits demonstrated variable azole chemosensitizing activities depending on the tested <i>Candida</i> species and the azole drug. Pitavastatin displayed broad-spectrum synergistic interactions with both fluconazole and voriconazole against isolates of <i>C. albicans</i>, <i>C. glabrata</i>, and <i>C. auris</i>. Ospemifene was able to interact synergistically with itraconazole against multiple fungal isolates including <i>Candida</i>, <i>Cryptococcus</i>, and <i>Aspergillus</i> species. Sulfa drugs displayed potent synergistic activities with different azoles against <i>C. albicans</i>, however, a limited efficacy was observed against efflux-hyperactive isolates such as <i>C. auris</i>. On the other hand, both lopinavir and aprepitant exerted potent and broad-spectrum synergistic activities with itraconazole and were effective against multiple <i>Candida</i> species including <i>C. albicans</i>, <i>C. auris</i>, <i>C. glabrata</i>, <i>C. krusie</i>, <i>C. tropicalis</i>, and <i>C. parapsilosis</i>. Furthermore, using <i>Caenorhabditis elegans</i> as an infection model, all drug combinations significantly reduced the fungal burden in the infected nematodes and significantly prolonged their survival as compared to single-drug treatments. Multiple phenotypic and molecular assays indicted that the identified hit compounds use distinct mechanisms to enhance the antifungal activity of azole drugs. These mechanisms include efflux pump inhibition, interference with the folate biosynthesis and disturbance of iron homeostasis. Taken together, this study reveals novel and potent azole chemosensitizing agents effective against multiple azole-resistant isolates and opens the door for more investigations to assess their clinical potential in human medicine as promising antifungal adjuvants.</p>

<p><a href="" target="_blank">Synthetic microbial communities (SynComs) are an important focus in modern microbiology. SynComs are used for studying the dynamics of naturally occurring microbial communities ranging from soil to the human gut. Synthetic refers to the assemblage of some or all the members of these communities in a laboratory setting. SynComs allow for communities difficult to study <i>in situ</i> to be studied in a controlled environment, or they may be used to create beneficial products like biofertilizers. When SynComs are used as products, the focus shifts to optimizing a desired outcome of a culture. For biofertilizers, a high diversity is key to producing a functionally redundant product for stimulating plant growth. Media manipulation is a common approach for inducing community changes in a SynCom. Amino acids (AA) are a media supplement that soil microbes (often the components of biofertilizers) are particularly fond of to support their metabolic activities. This study took a scale-up approach to assess the changes in growth dynamics of a SynCom (Environoc© 401) when supplemented with different concentrations of commercially available AA products from plant and animal sources. Expanding from microplates, to shake flasks, then into a 4L bioreactor, Environoc© 401 cultures were compared for their maximum growth rate, time in lag phase, and final growth (optical density or viable cell density) when supplemented with these AA products at various concentrations. Furthermore, Illumina sequencing of the 16S rRNA gene was used to evaluate community-level changes from these treatments based on taxonomic, alpha (Chao 1 and Shannon indices), and beta diversity (Generalized UniFrac) of shake flask and bioreactor samples. Quantitative PCR was also used to assess the relative change of three select species of the SynCom in each AA treatment. Shake flask data revealed significant changes in the growth dynamics of the SynComs within AA treatment groups. Whether the AA was animal or plant derived, generally as the concentration of AA increased, the maximum growth rate decreased, lag time increased, and final growth readings increased. The best AA supplement and rate according to the growth metrics and <i>Curveball </i>analysis was Stimtide at a 33% supplementation rate. This was compared with the control at the bioreactor scale where it showed higher overall final growth and a higher taxonomic and alpha diversity. The scale-up approach to this study was successful at selecting the best amino acid supplement type and rate despite having less sophisticated control and monitoring compared to larger scales (i.e. the bioreactor). Use of the <i>Curveball</i> modeling program was useful for treatment group selection but did not always predict the outcomes seen in the live cultures. The use of AA as a media supplement can increase growth and diversity of a SynCom, though not all AA supplements or rates affect growth and community dynamics in the same way</a>.</p>

<p dir="ltr">This thesis explores the complex relationship between <i>Listeria</i> <i>monocytogenes</i> and food processing environments (FPEs), focusing on the persistence mechanisms of this pathogen despite rigorous sanitation efforts. <i>L</i>. <i>monocytogenes</i> is a significant public health concern due to its association with high mortality rates in vulnerable populations. Recent studies, including the first chapter of this thesis, have highlighted the challenges in eradicating this pathogen from FPEs, suggesting that biofilms play a crucial role in its persistence. Despite various strategies and enhanced sanitation protocols, the eradication of <i>L</i>. <i>monocytogenes</i> remains elusive, underlining the need for a deeper understanding of its biofilm-associated resilience. Chapter two synthesizes findings from a systematic review and meta-analysis of studies examining the microbial communities on FPE surfaces through metagenomics, aiming to uncover patterns that might explain <i>Listeria</i>'s long-term survival. In chapter three, the thesis delves into the nuanced role of biofilm composition and microbial diversity as factors enabling the persistence of <i>Listeria monocytogenes</i> in food processing environments (FPEs), despite comprehensive sanitation efforts. This section builds on the understanding that <i>L. monocytogenes</i> does not inherently form robust biofilms but thrives within complex microbial communities present on FPE surfaces. The findings presented in this thesis contribute to a more profound comprehension of <i>L. monocytogenes</i>' survival strategies, proposing a shift in focus towards the microbial ecosystem's metabolic interplays for the development of targeted control measures. This approach not only opens new avenues for research but also suggests practical implications for enhancing food safety protocols by considering the broader microbial dynamics at play within biofilms on FPE surfaces.</p>

The aim of this thesis was to identify the denitrifying microbial communities in activated sludge from full-scale treatment plants and from small-scale reactors exposed to acetate or methanol as external carbon sources. Biological denitrification is currently the most widely used, sustainable and cost-effective process to remove nitrogen from wastewater. Increasingly strict effluent discharge standards are posing significant challenges to plant operators to reduce effluent NO3--N concentrations to levels as low as 2-3 mg L-1 or even lower. The lack of sufficient influent carbon in many municipal wastewater treatment plants makes it very difficult to achieve such low NO3--N concentrations in the effluent. An effective solution to the problem is to introduce additional external carbon sources to enhance denitrification. The selection of external carbon sources is not purely based on costs but is also dependent on the possible microbial transformations that these carbon sources may bring about in activated sludge. The most common carbon source used is methanol due to its low cost, but it has been found to cause long delays until an improvement in denitrification performance is observed. On the other hand, acetate has been found to improve denitrification almost instantaneously when added, but it has a significantly higher cost. In this study, methanol and acetate utilising denitrifiers were investigated in activated sludge with and without enrichment in laboratory scale bioreactors. The relevant denitrifiers were identified and evaluated in situ using culture independent methods particularly stable isotope probing (SIP), 16S rDNA cloning, fluorescence in situ hybridisation (FISH) and microautoradiography (MAR). Activated sludge collected from a biological nutrient removal plant exhibiting good denitrification was enriched in an anoxically-operated sequencing batch reactor (SBR) by feeding methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was operated over a duration of 7 months and the SBR denitrification rate improved from 0.02 mg NO3--N mg mixed liquor volatile suspended solids (MLVSS)-1 h-1 to a steady-state value of 0.06 mg NO3-N mg MLVSS-1 h-1. At steady state operation the enriched biomass was subjected to SIP with 13C-methanol to biomark the denitrifiers capable of utilising methanol under anoxic conditions. The separated 12C-DNA and 13C-DNA fractions from the SIP experiment were individually subjected to full cycle rRNA analysis. The dominant 16S rRNA gene phylotype (Group-A clones) in the 13C-library was closely related to the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96-97% sequence identities), while the most abundant clone groups in the 12C-library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes were designed for FISH to target the Group-A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes on SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, no correlation was found between denitrification rate and the relative abundances of the well known denitrifying genera Hyphomicrobium and Paracoccus nor the Saprospiraceae-clones visualised by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C] methanol uptake in the enriched biomass. As observed in full-scale operations, the methanol-fed SBR experienced a lag period of several weeks before denitrification performance increased. Using FISH quantification, it was shown that this coincided with the lag phase in the growth of the DEN67-targeted denitrifying population. It was therefore concluded that the Methylophilales bacteria dominant in our SBR system are likely to be important in full-scale methanol-fed denitrifying sludges. The acetate utilising microbial consortium in activated sludge was investigated without prior enrichment using stable isotope probing (SIP). 13C-acetate was used in SIP to biomark the DNA of the denitrifiers. The extracted 13C-DNA fraction was subjected to a full cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C-library were closely related to bacterial families Comamonadaceae and Rhodocyclaceae of class Betaproteobacteria (96-97% sequence identities). Seven oligonucleotide probes (DEN444, DEN220, DEN581, DEN441, DEN124, DEN220a and DEN1454) for use in FISH was designed to specifically target the identified phylotypes. Application of these probes on the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated over a duration of 16 days indicated a strong correlation between the level of CFDSBR denitrification and relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH combined with microautoradiography (FISH-MAR) further confirmed that the DEN581- and DEN124-targeted cells dominating the CFDSBR were capable of taking up [14C] acetate under anoxic conditions. The initial occurrence of the DEN444- and DEN1454-targeted bacteria and the final dominance of DEN581- and DEN124-targeted bacteria in the CFDSBR community were likely related to the changing in-reactor nitrite concentrations during the first few days of CFDSBR operation. Hence, the DEN444- and DEN1454-targeted bacteria were hypothesised to have low affinities for nitrite while DEN124- and DEN581-targeted bacteria have higher nitrite affinities. However, it was clear that all probe-targeted bacteria were denitrifiers capable of utilising acetate as a carbon source. The rapid increase in numbers of the probe-targeted organisms positively correlates with the immediate increase in denitrification rates. The rapid response and community shifts observed when acetate was used to enhance denitrification suggest that an intermittent application of acetate is quite effective to temporarily enhance the denitrification capacity of a treatment plant. However, the importance of a bacterial impact assessment of activated sludge subjected to intermittent acetate supplementation is recommended prior to the wide use of acetate in the wastewater industry. The acetate utilising denitrifying microbial communities investigated in the previous chapter were characterised according to their eco-physiological properties using the r- and K-selection criteria. The electron donor (acetate) and acceptor (nitrite) affinities of these probe-identified denitrifiers were used as traits for this characterisation. The substrate to microorganism (S/M) ratio was manipulated to provide high and low substrate concentrations in the reactor to create conditions favourable for r- and K-strategists, respectively. Two factors, namely feeding regimes and sludge retention times, were studied to achieve the desired S/M ratios and enable r/K characterisation. The high substrate affinities and high specific growth rates of two probe-identified denitrifiers (DEN124 and DEN581) did not enable resolution of these two organisms with the feeding regimes used in this study. However, the application of different sludge retention times as a control strategy to maintain constant high and low in-reactor S/M ratios enabled characterisation of the two probe-targeted denitrifiers DEN124 and DEN581 as K- and r-strategists, respectively. The in-reactor S/M ratios applied in this study did not facilitate the characterisation of populations targeted by probes DEN444 and DEN1454. The minor fluctuations of the S/M ratios during a cycle in the SBR operation was considered as a drawback, but conclusive results could still be obtained from the study. A chemostat reactor operation with constant loading and variable flow rates is suggested as an alternative. Conclusively, this study was able to identify specific groups of denitrifying microorganisms in activated sludge when exposed to acetate and methanol. Unlike most previous studies, which relied on culture dependent methods, this study adopted a pure culture independent approach to identify microorganisms in relation to their function, i.e. denitrification. Moreover, acetate denitrifiers were in situ characterised based on eco-physiological properties. The identification of denitrifying communities in this study has paved the way to a larger research project on the optimisation of denitrification processes with external acetate, methanol and other carbon supplements. As such, this study has contributed significantly to the understanding of the denitrification processes by linking process data with microbial investigations.