Mechanisms Involved in the Expression of the AmpC Beta-lactamase Gene blaCMY-2 in Escherichia coli

Enterobacteriaceae that express plasmid-encoded AmpC β-lactamases can be resistant to third generation cephalosporins and β-lactam/β-lactamase inhibitor combinations. Infections by these bacteria are associated with increased patient morbidity and mortality. Little is known about how plasmid-encoded AmpC genes are regulated or expressed. The experiments described in this dissertation sought to determine mechanisms that influence the transcription of blaCMY-2; the most common plasmid-encoded AmpC β-lactamase gene found in E. coli worldwide. Four clinical piperacillin/tazobactam-susceptible E. coli parent strains carrying blaCMY-2 and twelve piperacillin/tazobactam-resistant mutants selected from the parent strains were the focus of study. It was hypothesized that sequence changes upstream of blaCMY-2 were driving increased blaCMY-2 expression and enabling resistance to piperacillin/tazobactam in the mutants. It was also hypothesized that transcription factors binding to sequence upstream of blaCMY-2 promoter regions were influencing expression. Three important findings were made testing these hypotheses. 1) It was found that only 33% of the piperacillin/tazobactam-resistant mutants were overexpressing blaCMY-2. No upstream sequence changes were found in blaCMY-2 overexpressing mutant strains. For all overexpression mutants, changes in blaCMY-2 transcript level were associated with increased copy number of their blaCMY-2 encoded plasmid. Two mutants with blaCMY-2 on a 100 kb IncI1 plasmid had point mutations in the inc antisense RNA gene that controls IncI1 copy number. 2) Examination of upstream sequence for two parent strains identified a novel divergent tandem blaCMY-2 arrangement flanking an IS5 insertion element. Strains with this feature had 2-fold higher blaCMY-2 expression than a single arrangement strain. 3) The transcriptional activator Rob was identified binding sequence upstream of the distal blaCMY-2 promoter sequence within the insertion element ISEcp1. Additional studies indicated Rob was activated by β-lactam exposure and therefore may enhance blaCMY-2 expression when β-lactams are present. Further work will be needed to determine how gene copy number and Rob play a role in β-lactam resistance among clinical E. coli strains carrying blaCMY-2 .

Grantor dc:publisher

Creighton University

Year dc:date.issued

2013

Author dc:creator

• Kurpiel, Philip Marion

Advisor dc:contributor.advisor

• Hanson, Nancy D.

Statement dc:rights

• Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10504/47482

OAI identifier oai:identifier

oai:cdr.creighton.edu:10504/47482