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"Carbapenem Resistant Enterobacteriaceae: Hunting the Nightmare Bacteria"

by
Kalyan D. Chavda
Molecular Biology, Genetics & Cancer Program
M.Sc., 2009, University of Skövde, Sweden
B.Sc., 2007, Virani Science College (Saurashtra University), India




Thesis Advisor: Barry N. Kreiswirth, Ph.D.
Professor
Public Health Research Institute Center

Monday, May 1, 2017
12:00 PM, ICPH Auditorium


Abstract

Klebsiella pneumoniae carbapenemases (KPCs) were first identified in 1996 in the USA. Since then, regional outbreaks of KPC-producing K. pneumoniae have occurred in the USA, and have spread internationally. A particularly troubling aspect is that blaKPC is commonly harbored on conjugative plasmids, enhancing the capacity for horizontal transfer to other susceptible strains, as well as other bacterial genera. Therefore, unraveling the nature and genetic basis of KPC-harboring Enterobacteriaceae is critical to understanding the spread and acquisition of these resistance-determining elements. The focus of this thesis is to investigate the role of bacterial strains and plasmids in the spread and emergence of blaKPC among Enterobacteriaceae. We have previously characterized the genetic structure of the successfully disseminated K. pneumoniae clone sequence type 258 (ST258) and identified a recombination hotspot that accounts for the majority of divergence among ST258 clinical isolates. However, this thesis demonstrates that unlike K. pneumoniae, the spread of blaKPC in Escherichia coli is largely due to horizontal transfer of blaKPC-harboring plasmids and elements into diverse genetic backgrounds. Interestingly, multiple factors including acquisition of specific plasmids, successful host lineages, and plasmid-host synergy are driving the molecular evolution of carbapenem resistance within the genus Enterobacter. Augmenting this problem of resistance is an erroneous identification of Enterobacter strains because of ambiguous typing methods and imprecise taxonomy. In this study, we used a whole-genome-based comparative phylogenetic approach to revisit and redefine the genus Enterobacter. Previous studies in our laboratory suggested that dissemination of blaKPC among K. pneumoniae ST258 is largely associated with plasmids belonging to three major incompatibility groups: pKpQIL (IncFIIK2 group), IncI2, and, IncFIA. By contrast, blaKPC is harbored by different plasmid Inc groups in E. coli, including IncN, IncFIA, IncFIIK2, IncFIIK1, and two novel replicons). Remarkably, among Enterobacter spp. there is clonal spread of an E. xiangfengensis ST171 clone harboring blaKPC-3 on an IncFIA plasmid, and horizontal transfer of pKPC_UVA01-like plasmids in multiple phylogenomic groups. Notably, there are common blaKPC-harboring plasmids in K. pneumoniae that are nearly absent in Enterobacter spp. In order to have rapid and accurate resistance information, we developed an Ion Torrent NGS platform to detect the most prevalent -lactamases in a comprehensive and clinically relevant assay. In addition, we developed multiplexed molecular beacon probes in a real-time PCR assay to identify prominent extended-spectrum--lactamase, plasmid-mediated AmpC -lactamase and carbapenemase genes directly from perianal swab specimens. The work presented here identifies the diverse mechanisms, and improves our understanding of the factors that drive the molecular evolution of carbapenem resistance in Enterobacteriaceae. The findings in this thesis provide a solid genomic framework that will serve as an important resource in future development of molecular diagnostics, and in supporting drug discovery programs.


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