Theses and Dissertations from UMD
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Item AN INVESTIGATION ON THE MOLECULAR BASIS FOR DIMER FORMATION OF A BACTERIOPHAGE ENDOLYSIN POSSESSING ANTIMICROBIAL ACTIVITY AGAINST STREPTOCOCCUS PNEUMONIAE(2023) Alreja, Adit Bipin; Nelson, Daniel C; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The global rise of antibiotic resistance casts a shadow on treating infectious disease. An alternative to the use of antibiotics is bacteriophage-derived peptidoglycan hydrolases called endolysins. Endolysins, produced at the end of a bacteriophage replication cycle, cause bacterial cell lysis and virion release. When applied exogenously as recombinant proteins, they are also capable of cleaving the Gram-positive bacterial peptidoglycan. Various studies conducted in vitro and in vivo showcase the therapeutic potential of endolysins as the next generation of antimicrobials. Streptococcus pneumoniae is the most common cause of a variety of infections ranging from otitis media to invasive bloodstream infection (bacteremia) and meningitis (brain infection). While pneumococcal vaccination programs have proven to be effective, the high rates of antibiotic resistance reported for S. pneumoniae has led to the CDC classifying it as a “serious” threat. One of the most studied endolysins targeting S. pneumoniae is Cpl-1. This thesis represents an investigation into the molecular basis for dimer formation of the Cpl-1 endolysin which displays antibacterial activity against S. pneumoniae. In addition to disproving a long-accepted mechanism of dimerization of Cpl-1 in the presence of choline, we have conclusively identified the residue involved in the formation of the Cpl-1 dimer. Our findings led to the discovery of a novel C-terminal consensus sequence shared by all pneumococcal endolysins that informs their propensity to form dimers in the presence of choline. Next, through a bioinformatics approach we identified a new endolysin containing this C-terminal consensus sequence, produced it, named it SP-CHAP, and showed that it forms a dimer in the presence of choline, indicative of the widespread dimerization phenomenon associated with pneumococcal endolysins. Of interest, SP-CHAP is the first endolysin with antimicrobial activity against S. pneumoniae that possesses a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain. SP-CHAP was subsequently characterized for its biochemical and antimicrobial properties and benchmarked against Cpl-1. SP-CHAP is active in all physiologically relevant conditions (pH, temperature) against various S. pneumoniae strains and displays no activity towards oral/nasal commensal organisms. This enzyme also displays pneumococcal biofilm eradication ability to a greater extent than Cpl-1, as visualized by confocal microscopy. To further translate the antimicrobial potential of this enzyme, the antimicrobial efficacy of SP-CHAP was validated in a S. pneumoniae mouse nasopharyngeal colonization model. Our results demonstrate the therapeutic potential of SP-CHAP as an attractive endolysin to treat S. pneumoniae infections and warrant further translational development of this enzyme.Item HEATED RESISTANCE: THERMAL TREATMENT TECHNOLOGY MITIGATION OF BIOLOGICAL WASTES’ ANTIBIOTIC RESISTANCE AND GENE MOBILITY IN WASTE SYSTEMS.(2023) Poindexter , Carlton; Lansing, Stephanie; Environmental Science and Technology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The burgeoning global threat of antimicrobial resistance (AMR) has policy makers,veterinarians, farmers, and physicians re-evaluating antibiotic stewardship. Worldwide, millions of people are affected by multidrug resistant bacteria. Human and animal waste are primary transporters of antibiotics, antibiotic resistant bacteria (ARB), and antibiotic resistant genes (ARGs) through rural and urban systems. Resistance within biological waste, as it moves through the landscape and sanitary/manure infrastructure to adjacent natural systems, is yet to be fully understood. The various environmental conditions, bacterial composition, and genetic factors result in highly complex interdependent relationships that influence the occurrence and dissemination of ARB and ARGs. Understanding the fate of ARB and movement of ARGs is critical to evaluating environmental and anthropogenic impact. Agricultural systems and wastewater treatment plants are target locations for quantifying connections between clinical and animal antibiotic use and environmental AMR. Waste management techniques/technologies, such as compositing and anerobic digestion (AD), have been shown to be effective in combating AMR. Studies have highlighted temperature as a key environmental determinant that could influence antibiotic degradation, ARG, and ARB abundance. The proposed research examines advanced heat-based manure and wastewater technological capacity for AMR reduction, while measuring treatment impact on ARG dissemination. Focusing on the reduction of AMR within biological waste treatment and the distribution of AMR factors into the environment. A key metric to understanding AMR is accurate detection and quantitation of antibiotic concentration within manure and other biosolid waste products. The first phase of this dissertation research focused on the development of a liquid chromatography in tandem with mass spectrometry (LC-MS/MS) method for detecting multi-class antibiotics residuals in various manure substrates. To combat the challenges of manure heterogeneity, this work focused on novel extraction methodology to achieve higher recovery of tetracyclines, macrolides, sulfonamides, and beta lactams simultaneously in a complex manure matrix. The method includes a two-step, liquid-solid extraction using 10 mL of 0.1 M EDTA-McIlviane buffer followed by 10 mL of methanol. Reporting total antibiotic recoveries of 67–131% for tetracyclines, 56% for sulfonamide, 49–53% for macrolides, and 1.3–66% for β-lactams. This method is novel in its application to four different manure substrate and utilization for waste risk assessment. This developed method was used for antibiotic quantification throughout the three thermal treatment studies to determine antibiotic concentrations, degradation, and monitor agricultural contributions to environmental AMR. The following research extensively focused on the evaluation of three advanced, high temperature waste treatment technologies on the mitigation of antibiotic resistance factors, including a composting rotary drum bedding recovery unit (BRU), thermophilic (55°C) and mesophilic (35°C) AD, and thermal hydrolysis pretreatment to reduce antibiotics, ARGs, and ARB. The assessment of environmental components, such as metals, bacterial community, and nutrient composition, are also included to determine any relational trends. The BRU study was conducted as a mass balance analysis to highlight antibiotics, ARGs and ARB partitioning within the BRU system. Dairy manure samples were collected over 24-hour period as the manure was treated with a solid-liquid separator producing two streams of substrates (liquids and separated solid), with the separated solid fraction continuing to the high temperature BRU processing. This study generated a mass flow analysis of manure and partitioning of antibiotic resistance factors throughout the manure treatment system. The study indicated that most of the manure mass containing the AMR factors goes untreated following solid-liquid separation, with 95% of the mass pumped to a storage lagoon and 5% proceeding to BRU processing. The removal of antibiotic residuals during BRU processes was insignificant, yet the BRS processing was 100% effective in removing the ARB examined. Five (Intl1, sul1, tetQ, tetX and tetM) of the eight ARGs were found to have significant reduction (>95%) following the thermophilic rotary drum composting portion of the BRU system. While the three other ARGs (tetW, ermB and bla2) remained constant despite treatment. An AD experiment was implemented as lab-scale destructive assay, highlighting antibiotic removal at two temperature and over time. This destructive batch assay used 18 sets of triplicate AD reactors filled with antibiotic spiked dairy manure and incubated under anerobic conditions at 35°C or 55°C for 43 days. Triplicates bottles destructively sampled at six time points (Day 0, 3, 9, 21, 36, and 43) to generate a degradation curve. The antibiotic erythromycin was more efficiently degraded under mesophilic conditions, with 100% removal by Day 36 compared to 97% reduction for thermophilic conditions during the 43-day digestion period. Though the higher temperature conditions proved better for oxytetracycline degradation, with 66% removal compared to only 22% removal for mesophilic conditions. ARG removal was dependent on the bacterial community, as the different conditions selected for various bacteria. While both conditions proved to be effective in reducing most of the ARGs (4-5 out of 8 genes tested), enrichment of other resistance genes was also documented. The tetW gene was found to increase >81% for both digester temperatures, highlighting the variety of bacteria harboring resistance genes and their varied responses to environmental conditions. The ermB genes was found to be located on the intl1 mobile genetic element and likely resided within bacteria that was not heat tolerant. This study highlighted the role of residential digester bacteria in harboring and potentially transferring resistance genes. The thermal hydrolysis (THP) technology ability to extensively lysis substrates was examined with subsequent AD for its impact on reducing antibiotic resistance factors. Comparative analysis of THP processing on spiked diary manure and wastewater biosolids followed by mesophilic digestion at 35°C was conducted to document substrate response to the treatment. AD was conducted as a destructive assay for 30 days with a 4-point sample curve (Day 0, 10, 20 and 30). This study can be found in the appendix. In addition to the lab and field work described above, this body of research also included a review and a proposed communication model for antibiotic resistance education for the general public. Lay audiences’ exposure and understanding of complex natural issues, such as AMR and climate change, are essential to behavioral changes and potentially legislative actions. By surveying and evaluating various aspects of scientific communication, this research empathized five rhetorical elements of storytelling shown to influence audience reception to scientific messaging. Communication techniques, such as narrative structure, normalization of the subject using human scaling, non-agentive language, trusted experts for message delivery, and future simulation, were all analyzed and reviewed for their effectiveness and incorporated into a mock model for presenting information about AMR. Bridging gaps between research institutions and the public is key to generating more inclusive spaces for innovation and mitigating issues interwoven within the built and natural environment.Item ENABLING RAPID PHENOTYPIC DETECTION OF CEPHALOSPORIN RESISTANCE BEYOND THE CENTRAL LABORATORY(2019) Nguyen, Hieu Thuong; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The so-called bacterial “superbugs” are largely resistant to some of the most commonly prescribed antibiotics, including a drug class known as cephalosporins used to treat many hospital and community-acquired infections. This major public health threat has been acknowledged for decades by the Centers for Disease Control (CDC) as a major concern; yet, the detection of superbugs has not been made routine since standard testing practices have been limited to specialized “central” laboratories with sophisticated yet bulky and expensive equipment and highly trained personnel. As a result, the lack of simpler testing methods that can be used in everyday clinics and doctor’s offices can be viewed as a source of error contributing to incorrect antibiotic treatment and poorer patient outcomes, factors that drive even more advanced resistance, depleting our drugs or last resort. In this dissertation, we explore new strategies for simplified methods to test for cephalosporin resistance in order to give higher accessibility in the timely detection of superbugs to support the improvement of patient care. To do this, we take an organic chemistry and biochemical approach to develop new detection molecules that report resistance activity in bacteria expressing extended-spectrum β-lactamase (ESBL) enzymes, one of the most prolific resistance strategies used by superbugs. Next, we describe methods of integrating these detection molecules into practical testing methods, and detail the engineering of simpler assays that allow for rapid readout of ESBL phenotypes using commonplace laboratory plate readers, portable Raman devices, and even handheld personal glucose meters (used for diabetes monitoring) purchased from the drugstore.Item Structure-Guided Engineering of a Multimeric Bacteriophage-Encoded Endolysin PlyC(2019) Shang, Xiaoran; Nelson, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Emerging antibiotic resistance has become a global health threat. One alternative to antibiotics is bacteriophage-encoded endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage replication cycle resulting in bacterial cell lysis and progeny bacteriophage release. Endolysins are also capable of destroying the Gram-positive bacterial peptidoglycan when applied externally as recombinant proteins. These enzymes typically consist of an enzymatically active domain (EAD) and a separate cell wall binding domain (CBD). Studies have shown therapeutic efficacy of endolysins in vitro and in vivo, with no resistance developed to date. An endolysin from the streptococcal C1 phage, known as PlyC, has the highest activity of any endolysin reported. It also has a unique multimeric structure consisting of one activity subunit (PlyCA) harboring two synergistically acting catalytic domains, GyH and CHAP, and eight identical binding subunits (PlyCB) forming an octameric ring. Groups A, C, and E streptococci as well as Streptococcus uberis are sensitive to the lytic activities of PlyC. In order to harness the potent activity of PlyC for use against other bacteria, we sought to change/extend the host range of PlyC by engineering PlyCB and PlyCA, respectively. We first used a structure-guided mutagenesis method to obtain the single PlyCB monomer subunit, PlyCBK40A E43A (PlyCBm), aiming to study the binding mechanism of PlyCB. Via fluorescence microscopy and binding assays, we determined that PlyCBm retained the host range of the octamer with a much lower binding affinity, which suggests the PlyCB octamer binds concurrently to a specific epitope on the bacterial surface resulting in a tight, stable interaction. Thus, it is not feasible to change/extend the PlyC host range via engineering PlyCB. Next, we proposed a novel design to engineer PlyCA. We successfully created two chimeric endolysins, ClyX-1 and ClyX-2, possessing the synergistic activity of the GyH and CHAP catalytic domains, but extended the host range to include, Streptococcus pneumoniae, Group B streptococci, Streptococcus mutans, and Enterococcus faecalis, all strains previously insensitive to PlyC. Finally, we tested a novel hypothesis that a positively charged catalytic domain could display lytic activity in a CBD-independent manner resulting in a broad host range. Using the PlyC CHAP domain as a model, we converted the net surface charge of the CHAP domain from negative three to positive one through positive seven. Notwithstanding the range of charges, our mutant CHAP domains did not show lytic activity in a CBD-independent manner, suggesting that other factors, like surface charge distribution, need to be considered in such a way of engineering.Item Assessing the Presence of Antibiotic-Resistant Enterococcus in Reclaimed Water Used for Spray Irrigation(2013) Carey, Stephanie Ann; Sapkota, Amy R; Turner, Paul; Maryland Institute for Applied Environmental Health; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Reclaiming wastewater is increasing in the US to combat dwindling freshwater supplies. This water potentially contains pathogenic bacteria; therefore, we evaluated the occurrence, concentration, and antimicrobial susceptibilities of Enterococcus spp.--an important opportunistic pathogen that remains a leading cause of nosocomial infections--in reclaimed water used for spray irrigation (SI). A total of 48 wastewater effluent and SI samples were collected in 2009 and 2010 from the Mid-Atlantic and Midwest regions of the US. Enterococci were isolated using membrane filtration, confirmed using biochemical tests and PCR, and tested for antimicrobial susceptibility using the Sensititre® dilution system. We detected total enterococci and vancomycin-resistant enterococci (VRE) in 68% (27/40) and 8% (3/40), respectively, of all SI samples. VRE and vancomycin-intermediate enterococci (VIE) represented 2% (1/41) and 10% (4/41), respectively, of the total enterococci recovered from all SI sites. Our findings show that SI workers may be exposed to enterococci during spray irrigation activities.Item ISOLATION, IDENTIFICATION, AND ANTIMICROBIAL SUSCEPTIBILITY ANALYSIS OF ENTEROCOCCCUS SPP. AND SALMONELLA SPP. FROM CONVENTIONAL POULTRY FARMS TRANSITIONING TO ORGANIC FARMING PRACTICES(2009) Kinney, Erinna Lea; Sapkota, Amy R.; Public and Community Health; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This baseline study evaluated prevalence and antibiotic resistance of food-borne bacteria as conventional poultry facilities transition to organic practices. Poultry litter, feed, soil, water samples and poultry questionnaire responses were collected from 10 conventional and 10 organic-transitioning poultry houses from March to June 2008. Enterococcus spp. (n=260) and Salmonella spp. (n=100) isolates were identified to species level and antimicrobial susceptibility testing was performed using the Sensititre® system. Statistical analyses were performed using STATA 10. Prevalence of Enterococcus spp. on organic-transitioning and conventional poultry farms was 100%; and prevalence of Salmonella spp. was 100% and 40%, respectively. Enterococcus isolates from conventional poultry houses displayed significantly higher percentages of resistance for 9 antibiotic agents compared to organic-transitioning isolates. Conversely, Salmonella spp. isolated from both conventional and organic-transitioning poultry houses exhibited similar antibiotic resistance patterns. Baseline findings suggest importance of poultry production practice in prevalence and antibiotic resistance patterns of food-borne bacteria.Item Programmable Biomolecule Assembly and Activity in Prepackaged BioMEMS(2008-10-21) Luo, Xiaolong; Rubloff, Gary W.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Antibiotic resistance is an increasing public health concern and few new drugs for bacterial pathogenesis have been obtained without addressing this resistance. Quorum sensing (QS) is a newly-discovered system mediated by extracellular chemical signals known as "autoinducers", which can coordinate population-scale changes in gene regulation when the number of cells reaches a "quorum" level. The capability to intercept and rewire the biosynthesis pathway of autoinduer-2 (AI-2), a universal chemical signaling molecule, opens the door to discover novel antimicrobial drugs that are able to bypass the antibiotic resistance. In this research, chitosan-mediated in situ biomolecule assembly has been demonstrated as a facile approach to direct the assembly of biological components into a prefabricated, systematically controlled bio-microelectromechanical system (bioMEMS). Our bioMEMS device enables post-fabricated, signal-guided assembly of labile biomolecules such as proteins and DNA onto localized inorganic surfaces inside microfluidic channels with spatial and temporal programmability. Particularly, the programmable assembly and enzymatic activity of the metabolic pathway enzyme Pfs, one of the two AI-2 synthases, have been demonstrated as an important step to reconstruct and interrogate the AI-2 synthesis pathway in the bioMEMS environment. Additionally, the bioMEMS has been optimized for studies of metabolic pathway enzymes by implementing a novel packaging technique and an experimental strategy to improve the signal-to-background ratio of the site-specific enzymatic reactions in the bioMEMS device. I envision that the demonstrated technologies represent a key step in progress toward a bioMEMS technology suitable to support metabolic engineering research and development.Item DETECTION AND CHARACTERIZATION OF ESCHERICHIA COLI O157:H7 AND SALMONELLA IN FOOD(2004-04-26) CUI, SHENGHUI; MENG, JIANGHONG; Food ScienceEscherichia coli O157:H7 and Salmonella are among the most important foodborne pathogens that cause millions cases of infections and hundreds deaths each year in the United States. Beef and poultry products are frequently recognized transmission media for these two organisms. Rapid detection and isolation methods applied to beef or chicken products are expected for these two bacteria. A rapid sample preparation method for E. coli O157:H7 detection by PCR method in ground beef samples was developed by combining different techniques, including filtration, centrifugation, enzyme digestion, and DNA extraction. The detection limit of this method was 103 cells/g without enrichment, and 100 cells/g can be detected after 6 h en-richment. For Salmonella, a poultry specific isolation method was modified from the USDA/FSIS manual by considering the specific characteristics of poultry products. Higher than 95% of the suspect colonies isolated by using the modified method were confirmed as Salmonella by PCR/API 20 E tests. This method was applied on retail organic and conventional chicken samples for Salmonella isolation. All Salmonella isolates were further characterized by serotyping, PFGE and antibiotics susceptibility tests. The results indicated that organic and conventional chicken samples were frequently contaminated with Salmonella, and that Salmonella from organic chicken were more susceptible to antimicrobials commonly used in human and veterinary medicine. High acid resistance capability is another unique characteristic of E. coli O157:H7 which is correlated with low infection dose of this pathogen. Survival mechanism of E. coli O157:H7 cells in gastric juice or acidified LB (pH 2.5) was studied, it was found that the limited availability of glutamate and/or arginine creates an illusion of cell-density-dependent acid sensitive phenotype of E. coli O157:H7 during acid-challenge.