Rohan Shah

• Many antibiotics have intracellular targets and their pharmacological action is well characterized. However, the antibiotic uptake mechanism in bacteria is not fully resolved. Therefore, a greater efforts are required to understand the uptake of these drugs across the outer membrane in order to counteract bacterial resistance. A robust and sensitive technique like mass spectrometry can effectively be used to detect drug accumulation in bacterial cell. In a nutshell, the first part of research will be focused on the method to detect antibiotics in bacterial cell and the second part, will be focused on quantitative number of active influx porin in bacterial mutants. The first part of this thesis provides insight on how mass spectrometry can be used to study intracellular accumulation of antibiotics in Gram-negative bacteria. In this study, a high-throughput method using UHPLC-ESI-QTOF/MS was developed and different antibiotics are (Ciprofloxacin, Levofloxacin, Tetracycline, Linzolide, Fleroxacin and addition of other efflux inhibitors) used to measure intracellular accumulation in Escherichia coli. The different mutants of W3110 E. coli, wild-type, acrAB efflux pump KO,ompFompC double porin KO, and ompFompCacrAB triple mutants were used to study differential drug uptake in bacteria. Where the efflux mutant showed two-fold higher antibiotic accumulation when compared to wild-type and double porin mutant. There is an alternative uptake pathway expressed in double porin mutant due to which antibiotic accumulation is observed. Thus, this methodology could be implemented in study of xenobiotics uptake as a strategy to complement the knowledge of the dietary composition in foodomics (Improvement in food and nutrition through process of technology). The second part explains the importance of porins in E. coli and sheds light on correlation of porin regulation between different mutant strains. The study is well understood by using isotope labeled peptide in quantification.