Abstract
Klebsiella pneumoniae and Escherichia coli are major nosocomial pathogens. A clinical isolate of K.pneumoniae and E. coli were found to produce a novel plasmid-encoded ampC P-lactamase gene, blaFOX-5b- These isolates conferred high level resistance to a broad spectrum of antibiotics, including third generation cephalosporins and cephamycins. Expression of the structural gene of blaFOX-s from a heterologous promotor does not confer resistance to this spectrum of P-lactam drugs. It was hypothesized that promoter elements located upstream of blaFOX-5b within the plasmid sequence were required for high level expression to confer resistance. Therefore to address the hypothesis of this thesis research, the promoter of a novel plasmid-encoded P-lactamase gene, blaFOx-5b was located and the genetic elements responsible for conferring high-level resistance to cefoxitin was determined. A GenomeWalker kit was used to obtain a 1.5 Kb fragment upstream of the blaFOX-5b structural gene from a clinical isolate of K. pneumoniae. Sequence analysis of the 1.5 Kb fragment revealed the entire insertion sequence (IS) of ISAS2 from Aeromonas salmonicida with 98% similarity. Primer extension analysis was used to map the start site of transcription and determine the number of initiated transcripts expressed in the clinical isolates. Four deletion clones were constructed using PCR and represent the entire h/aFOx-5b structural gene plus regions corresponding to 4, 45, 107, 262, 437 or 593 base pairs upstream of the translational start codon. E. coli transformants of the deletion clones with and without the -35 region had cefoxitin MICs >256 ug/ml and 6 pg/rnl, respectively. Copy number was determined using PCR analysis. The low copy number (2) of this plasmid suggests cefoxitin resistance in these isolates is mainly due to expression of the blaFOX-5b P-lactamase and not copy number.