Abstract

An estimated 70% of bacterial infections in the United States are resistant to first-line antibiotics. Of these, among the most difficult to treat are highly resistant to carbapenems, an antibiotic class used as a last resort. Septicemia, particularly when caused by a carbapenem-resistant organism, is among the most challenging clinical scenarios to treat, with an overall mortality rate of carbapenem-resistant septicemias 63.8%. Encouragingly, if effective antimicrobial treatment begins within 1-3 hours of septic shock onset, patient survival rates are around 80%. The first section of this work describes a real-time PCR-based assay that when coupled with existing bacteria-blood separation technology, can rapidly identify genes present in a multi-drug resistant bacterial sample at physiologically significant levels (<10 CFU/mL). Primers and probes were designed which identify all subtypes of the most common carbapenemase genes in carbapenem-resistant infections in the United States: Klebsiella pneumoniae Carbapenemase (KPC); New Delhi Metallo-beta-lactamase (NDM); Cefotaximase-Munich (CTX); Cephamycin AmpC beta-lactamase (CMY); and Oxacillinase-48 (OXA-48). The information provided by this assay will supply physicians with critical drug resistance information within two hours of septicemia onset and allow for timely prescription of effective antimicrobials which correspond to the resistance gene(s) present in the causative organism. Increased understanding of the mechanisms and evolution of carbapenem resistance; in particular, the phenomenon of heteroresistance, is of significant clinical import. As such, the second section of this work examines a clinical, carbapenem-resistant E. coli isolate that when cultured in the prolonged absence of antibiotic pressure, achieves clinically relevant levels of susceptibility to imipenem and doripenem through loss of its blaNDM-1-harboring transposon at the DNA level. Through full genomic sequencing of the highly resistant population, we theorize that the resistance level was achieved through loss of genomic regions whose maintenance either exerted fitness costs in the presence of doripenem, or which encoded genes whose expression inhibited doripenem resistance via pathways that influence outer membrane porin expression. Along with the disappearance and resurgence of carbapenem resistance, emergence of a highly carbapenem-heteroresistant population occurred early and remained throughout the duration of the study.

Degree

PhD

College and Department

Life Sciences; Microbiology and Molecular Biology

Rights

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