Development of Rapid Testing Technique to Diagnose Antibiotic Resistance

Abstract

Antibiotic resistance is a rapidly growing global health concern, creating an urgent need for detection methods that are both rapid and reliable. Antibiotic resistance occurs when microorganisms survive or grow despite exposure to antibiotic drugs designed to kill or inhibit them. It happens due to several factors like inappropriate prescribing, overuse, misuse, antibiotic residue intake from food items like eggs, meats, vegetables. Recent data shows, approximately 1.27 million deaths occurring each year, and the figure could increase to 39 million by 2050 if failed to take action. Besides, the overuse and misuse of antibiotics have led to the rise of multidrug-resistant organisms, commonly known as "superbugs," which are resistant to multiple antibiotics and complicate the treatment. The economic impact is also alarming. World Bank is projecting potential global GDP losses of up to $3.4 trillion annually by 2030 due to the antibiotic resistance. Traditional methods for detecting antibiotic resistance primarily involve phenotypic tests like disk diffusion, broth/ agar dilution, e-test and some automated systems that observe the growth response of bacteria to antibiotics. Though traditional methods are widely used for detecting antibiotic resistance but it has several drawbacks like time-consuming, labor-intensive, variability in data or results. The main challenge for the doctors is getting test data prior to prescribe right antibiotics to the patient. Considering the patient situation, most of the cases doctor prescribe antibiotic based on the assumption. This doctoral research introduces an electrochemical sensing strategy for the detection of antibiotics and the assessment of resistance phenomena, built upon a poly-L-glutamic acid (PGA) modified glassy carbon electrode (GCE). The objective is to seek a rapid and reliable detection mechanism of antibiotic resistance. Aligning with the research objectives, the work is divided into four experimental phases, each addressing a distinct objective in sensor development and application like antibiotic determination and evaluation of resistance phenomena. In the first phase, the glassy carbon electrode surface was modified through electropolymerization of L-glutamic acid to form a PGA coating. The modification was confirmed through a combination of analytical techniques. Cyclic Voltammetry (CV) demonstrated enhanced redox activity of the PGA modified GCE as compared to the bare GCE. Infrared (IR) spectroscopy revealed characteristic peak shifts consistent with polymer formation. Electrochemical Impedance Spectroscopy (EIS) showed reduced charge transfer resistance and improved sensitivity in Nyquist plots. Scanning Electron Microscopy (SEM) visualized a uniform polymer layer with increased surface roughness. These vi results verified the successful fabrication of PGA modified electrode with a highly responsive electrode surface. In the second phase of the study, we targeted to apply the PGA modified electrode to the detection of Ceftibuten, a β-lactam antibiotic. Versatile CV and Differential Pulse Voltammetry (DPV) were employed in phosphate buffer (pH 6.8), enabling trace-level detection of Ceftibuten. A model experiment for the detection of Ceftibuten was carried out; ie., in-vitro experiments with blood serum confirmed the method’s applicability in complex biological matrices. A simulation study incorporating β-lactamase enzyme demonstrated the sensor’s ability to detect antibiotic degradation, as indicated by a marked reduction in peak current corresponding to β-lactam ring cleavage. This will ultimately, facilitate to recognize the extend clinical study to determine the antibiotic content in blood or urine samples thus the detection of antibiotic resistance. The third phase was extended for the approach applying the same system to Cefuroxime, another β-lactam antibiotic, following the same methodological framework. The PGA-modified GCE again exhibited high sensitivity in both buffer and serum samples. Enzymatic degradation by β-lactamase produced a consistent decline in electrochemical signal, confirming the PGA modified GCE capability to monitor resistance-related biochemical changes. In the fourth phase, the PGA modified GCE was evaluated for Levofloxacin, a fluoroquinolone antibiotic. Trace detection was of Levofloxacin was achieved in buffer and serum as well as in a simulation study using Escherichia coli ATCC culture sample containing Levofloxacin revealed a significant drop in peak current after bacterial exposure, indicating detection of antibiotic interaction and potential resistance development for the antibiotics. This demonstrated a platform for the versatility for the detection of the resistance of both β-lactam and non-β-lactam antibiotics. Overall, the findings establish PGA-modified GCE-based electrochemical system as rapid, sensitive, and dependable tools for antibiotic detection and their resistance assessment. Unlike conventional microbiological assays, which often require more than 24 hours, this approach can deliver actionable results within 2-4 hours, offering a substantial advantage for timely clinical decision-making. The outcomes of this research provide a foundation for future development of point-of-care diagnostic systems and encourage further investigation across a wider range of antibiotic classes and resistance mechanisms.