The widespread emergence of antibiotic resistance determinants for nearly all drug classes threatens human health on a global scale. It is therefore essential to discover antibiotics with novel functions that are less likely to be influenced by pre-existing resistance mechanisms. An emerging approach to identify inhibitors of investigator-defined cellular processes involves screening compounds for antimicrobial activity under non-standard growth conditions. Indeed, by growing cells under conditions of stress, inhibitors of specific cellular targets can be enriched, thereby allowing for the identification of molecules with predictable activities in the complex environment of the cell. Here, I exploit cold stress in Escherichia coli to identify molecules targeting ribosome biogenesis and outer membrane biosynthesis. First, through a screen of 30,000 small molecules for growth inhibition exclusively at 15°C, I was able to identify the first small molecule inhibitor of bacterial ribosome biogenesis, lamotrigine. Second, by leveraging the idiosyncratic cold sensitivity of E. coli to vancomycin, I developed a novel screening technology designed to enrich for non-lethal inhibitors of Gram- negative outer membrane biosynthesis. From this platform, I identified pentamidine as an efficient outer membrane perturbant that was able to potentiate Gram-positive antibiotics against Gram-negative pathogens, similar to the polymyxins. Remarkably, however, this compound was able to overcome mcr-1 mediated polymyxin resistance. Together, this thesis highlights the utility of exploiting the bacterial cold stress response in antibiotic discovery. / Thesis / Doctor of Philosophy (PhD)

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This thesis explores a method to fabricate magnetic membranes with asymmetric distribution of particles and their testing as actuators. Focus of this research is to fabricate thin polymeric sheets and thickness range of 120-125µm, with asymmetric distribution of magnetic nano particles, employing micromagnets during the fabrication. The micromagnets are used to localize the magnetic particles during the curing process at selected locations. The effect of the asymmetric distribution of magnetic particles in the membrane is used for the first time. Magnetite (Fe3O4) is used as the magnetic particles that is embedded into a polymeric membrane made of polydimethylsiloxane (PDMS); the membrane is then tested in terms of deflection observed by using a high-resolution camera. From the perspective of the biomedical application, PDMS is chosen for its excellent biocompatibility and mechanical properties, and Fe3O4 for its non-toxic nature. Since magnetic actuation does not require onboard batteries or other power systems, it is very convenient to use in embedded devices or where the access is made difficult. A comparative study of membranes with asymmetric and randomly distributed particles is carried out in this thesis. The asymmetric distribution of magnetic particles can benefit applications involving localized and targeted treatments and precision medicine.

Biofouling represents the main problem in membrane filtration systems. Biofouling arises when the biomass growth negatively impacts the membrane performance parameters (i.e. flux decrease and feed channel pressure drop). Most of the available techniques for characterization of biofouling involve membrane autopsies, providing information ex-situ destructively at the end of the process. OCT, is non-invasive imaging technique, able to acquire scans in-situ and non-destructively. The objective of this study was to evaluate the suitability of OCT as in-situ and non-destructive tool to gain a better understanding of biofouling behavior in membrane filtration systems. The OCT was employed to study the fouling behavior in two different membrane configurations: (i) submerged flat sheet membrane and (ii) spacer filled channel. Through the on-line acquisition of OCT scans and the study of the biomass morphology, it was possible to relate the impact of the fouling on the membrane performance. The on-line monitoring of biofilm formation on a flat sheet membrane was conducted in a gravity-driven submerged membrane bioreactor (SMBR) for 43 d. Four different phases were observed linking the variations in permeate flux with changes in biofilm morphology. Furthermore, the biofilm morphology was used in computational fluid dynamics (CFD) simulation to better understand the role of biofilm structure on the filtration mechanisms. The time-resolved OCT analysis was employed to study the biofouling development at the early stage. Membrane coverage and average biofouling layer thickness were found to be linearly correlated with the permeate flux pattern. An integrated characterization methodology was employed to characterize the fouling on a flat sheet membrane, involving the use of OCT as first step followed by membrane autopsies, revealing the presence of a homogeneous layer on the surface. In a spacer filled channel a 3D OCT time series analysis of biomass development under representative conditions for a spiral-wound membrane element was performed. Biomass accumulation was stronger on the feed spacer during the early stage, impacting the feed channel pressure drop more than the permeate flux. OCT biofilm thickness map was presented as new tool to evaluate the biofouling development in membrane filtration systems through the use of a false color scale.

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