Intracellular genetic network involving anti-microbial resistance expression in Klebsiella pneumoniae strains which acquire carbapenem resistance in-vivo and in-vitro

Abstract

Antibiotic resistance is an important health crisis worldwide. It is predicted that by the year 2050, the number of deaths due to antimicrobial resistant bacteria is going to reach an all-time high of 10 million a year. The magnitude of impact of multi-drug resistant bacteria on lives of people has forced us to find novel drug targets and mechanisms to control these pathogens at the earliest. The pace of resistance acquisition by the bacteria has surpassed that of finding newer antibiotics. Klebsiella pneumoniae, an opportunistic pathogen, is well-known for its nosocomial pathogenicity by displaying resistance to most antibiotics commercially available, thereby, limiting treatment options. The aim of my dissertation was to find novel resistance mechanisms in clonally related carbapenemase non-producing carbapenem-resistant K. pneumoniae strains obtained from a patient after meropenem treatment. In addition, finding a reliable method for porin detection in carbapenem-resistant K. pneumoniae strains. Chapter I provides a brief overview on the history of antibiotics, antibiotic drug targets and resistance mechanisms, along with β-lactams and mechanisms of carbapenem resistance in K. pneumoniae. Chapter II describes finding the cause of resistance in a carbapenemase non-producing carbapenem-resistant K. pneumoniae clinical isolate that showed strong three dimensional bioassay test positive indicating the presence of carbapenemase enzyme. Radiation mediated mutagenesis was used to render the resistant strain susceptible. The cause of positive 3D bioassay was attributed to the presence of blaCMY-10 gene in the plasmid of the isolate which is an AmpC β-lactamase gene. Complementation of blaCMY-10 gene into clinical isolates and outer membrane protein (OMP) mutants concluded that the carbapenem resistance occurrence in blaCMY-10-carrying K. pneumoniae isolates was due to the loss of both OmpK35 and OmpK36 porins. Chapter III illustrates novel mechanisms that bring about meropenem susceptibility in K. pneumoniae acquired carbapenem resistance in-vivo and in-vitro. The putative candidate genes were short-listed using whole genome analysis, transcriptome analysis and a functional gene network called KlebNet. Complementation of KPHS_33600 (MFS transporter) and KPHS_46730 (garL) genes showed decrease in meropenem MIC (from ≥32 µg/ml to 8 µg/ml) in in-vivo resistant strain K56, and in-vitro resistant strain K26M, respectively. The complemented strains did not show reduction in fitness when grown in LB broth and Galleria mellonella larvae successfully recovered when treated with meropenem when infected with the KPHS_33600 and garL complemented strains. Possible mechanism of action has also been illustrated using transcriptome data obtained from the complemented strains. Chapter IV compares the use of different methods for OMP detection i.e. MALDI-TOF MS, SDS-PAGE, WGS and transcriptome data analysis. At present, SDS-PAGE is the gold standard for OMP detection. We found discrepancy in OMP detection using SDS-PAGE in K. pneumoniae, therefore, we compared the results using the methods mentioned above. In addition, peptide sequencing was carried out to confirm the SDS-PAGE bands. OmpK35 could not be detected using SDS-PAGE and MALDI-TOF MS. However, the results obtained from both these methods were identical, concluding that MALDI-TOF MS can replace SDS-PAGE. RNA analysis could not accurately confirm due to lower level expression of mutated genes. Whole genome and/or PCR followed by Sanger sequencing could accurately detect the OMPs, thereby making them the most reliable methods for OMP detection of K. pneumoniae clinical isolates. In conclusion, this dissertation is focused on finding resistance mechanisms, cause of resistances and reliable detection method for OMPs in carbapenem-resistant K. pneumoniae clinical isolates. Since K. pneumoniae is not very well studied as Escherichia coli or PAO-1 of Pseudomonas aeruginosa, there are several uncharacterized genes that are of research interest. Characterization of KPHS_33600, one of such uncharacterized transporter gene, can provide further insight into its functional relationship with meropenem susceptibility.