Rapid diagnosis of bacterial infections based on antibiotic ligands

Abstract

Throughout history, Epidemics and infectious diseases had impacted the person and human society. Before the discovery of infectious bacteria and antibiotics, humans did not understand and died by various infections and epidemics caused by infectious bacteria. In the 19th century, Infectious bacteria were found as the cause of infection and epidemic, and antibiotics began to be synthesized which were dramatically increased human health and population. However, infectious bacteria getting acquired antibiotic resistance with the overuse and abuse of antibiotics for all infections. In a 2013 CDC report for antibiotic resistance, in order to respond to this increasing antibiotic resistance, I need to develop new antibiotics and to prevent abuse of antibiotics to suppress the antibiotic-resistant infection. The abuse and overuse of antibiotics in hospitals started with the limitation of the conventional standard diagnostic method for infectious bacteria, which was time-consuming, or the opportunity of the false-positive signals. In this study, I present a more rapid and accurate diagnosis system to discriminate and detect infectious bacteria. In chapter 1, I introduce the history and diagnostics techniques of infectious disease and antibiotics. In chapter 2, colistin sulfate (polymyxin E) was used to label and diagnosis Gram-negative bacteria as a targeting ligand. Colistin is an antibiotic that forms pore on lipopolysaccharide (LPS) presented on the cell wall of Gram-negative bacteria, and the possibility of specific labeling properties on Gram-negative bacteria was confirmed through previous studies. To confirm and optimize the specific condition for diagnosis with colistin as a targeting ligand, I prepared fluorophore-conjugated colistin. The time and concentration to label Gram-negative bacteria were confirmed, and also prepared this system labeled Gram-negative bacteria specifically among various pathogens, cells, and biological components. In chapter 3, electrochemical sensors to discriminate Gram-positive and Gram-negative bacteria were assessed using colistin, as a Gram-negative specific ligand, and vancomycin, as a Gram-positive specific ligand. I synthesized and prepared antibiotic conjugated polymer dot, and coated silicon wafer with polymer dot solution to prepare electrochemical sensors. These electrochemical sensors were able to specifically identify and diagnose Gram-positive bacteria and Gram-negative bacteria, and furthermore, clinical samples from infected patients were also distinguished precisely. In addition, antibiotic resistance bacteria were confirmed with a shorter operating time than conventional methods, such as e-test or disk diffusion method, by using additional antibiotics in labeling solution. In chapter 4, I proposed two studies to measure antibiotic resistance via click chemistry and microfluidic channels. As a first trial, L-captopril, known as an inhibitor that blocks the active sites of NDM-1 which is a type of carbapenem-resistant Enterobacteriaceae (CRE), was attempted to label NDM-1 through a biorthogonal reaction. Since L-captopril is a molecule that strongly interacts with the Zn ion in the active site of NDM-1, L-captopril derivatives (Cap-ED-TOC and Cap-PEG-TCO) were prepared to label and inhibit NDM-1. Through the inhibition of antibiotic hydrolysis of NDM-1, I confirmed the labeling of NDM-1 with L-captopril derivatives. Afterward, Tz-fluorophore was introduced to react with captopril derivatives on NDM-1, and fluorescence signal would be obtained from NDM-1. However, after the second labeling reaction, the L-captopril derivative failed to label NDM-1 and no significant fluorescence signal was obtained. As a second trial, microbeads and microfluidic channels were used to confirm the antibiotic susceptibility of infectious bacteria or to accurately measure the minimum inhibitory concentration (MIC). In this study, magnetic beads helped to capture, concentrate, and isolate infectious bacteria from clinical samples, and the bacteria were inoculated into a microfluidic channel for gradient antibiotic susceptibility testing that maintains a gradient antibiotic concentration (GAST microfluidic channel). This GAST microfluidic channel can control and maintain the distribution of antibiotics, and also can grow inoculated bacteria. By controlling the distribution of antibiotics in GSAT microfluidic channel, GSAT conducted the antibiotic susceptibility test or measure more sensitive MIC with narrow antibiotic concentration. This study intended to help enable the use of intermediated-resistance antibiotics through more sensitive MIC measurements, and expand the available antibiotics in practical use.