Beta Lactam

Beta-lactam resistance

The early 1990s saw a rapid emergence of resistance to b-lactam antibiotics in P. aeruginosa isolates from cystic fibrosis patients due to in vivo selection of constitutive beta-lactamase producing strains that contribute to both morbidity and mortality in patients (1, 2, 3). The genetic events underlying the overproduction of this enzyme have not been elucidated. Before embarking on determining the various mechanisms that may contribute to enhanced expression of beta-lactamase levels in clinical isolates, we need to decipher how the genes involved in beta-lactamase production are normally regulated beginning with clues from what is known in other organisms. Very little is known about amp genes in general, and specifically, we lack any understanding of how these genes are regulated in P. aeruginosa. To date, it appears that beta-lactamase expression in Enterobacteriacea involves at least three loci containing five genes: ampC, ampR, ampD, and ampG, encoding the enzyme, positive regulator, negative regulator (an amidase), and a permease, respectively. The fifth gene, ampE has no known function. The objective of this research is to use genetic and molecular biological techniques and biofilm chambers to investigate the regulation of Sigma-lactamase genes in P. aeruginosa strains and to understand the mechanism used to develop resistance to this antibiotic. This knowledge will help us in the design of proper prophylactic and therapeutic treatments for P. aeruginosa infections.

Specifically this research will systematically analyze the amp genes in P. aeruginosa by conducting the following investigations: (a) Determine whether the genes, ampC, ampR, ampD, ampE and ampG are regulated at the level of transcription in the presence and absence of beta-lactamase inducers; (b) Determine whether the ampC, ampR, ampD, ampE and ampG genes regulate themselves and each other by using systematic mutagenesis of the amp genes, and analysis of the amp gene expression levels in the mutant cells; (c) Determine the expression levels of the amp genes in cells growing in a biofilm, as it has been clearly documented that biofilm-growing cells are extremely resistant to antibiotics; and (d) Determine if there are other beta-lactam responsive genes in addition to these amp genes and whether these genes regulate or participate in resistance to b-lactam antibiotics.

References Cited:

Chen, H. Y., M. Yuan, and D. M. Livermore. 1995. Mechanisms of resistance to beta-lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993. J. Med. Microbiol. 43:300-309.

Giwercman, B., P. A. Lambert, V. T. Rosdahl, G. H. Shand, and N. Høiby. 1990. Rapid emergence of resistance in Pseudomonas aeruginosa in cystic fibrosis patients due to in-vivo selection of stable partially derepressed beta-lactamase producing strains. J. Antimicrob. Chemother. 26:247-259.

Giwercman, B., C. Meyer, P. A. Lambert, C. Reinert, and N. Høiby. 1992. High-level beta-lactamase activity in sputum samples from cystic fibrosis patients during antipseudomonal treatment. Antimicrob. Agents Chemother. 36:71-76.

Mathee Publications

Jayawardena, S.R., K.F. Kong, S.D. Indulkar, A. del Puerto, C.L. Koh, N. Høiby and K. Mathee. Characterization of Pseudomonas aeruginosa AmpR that regulates expression of AmpC beta-lactamose and other virulence factors. Journal of Bacteriology (submitted)

A. Aguila. 2002. Topology analysis of Pseudomonas aeruginosa AmpG, a putative permease involved in beta-lactam antibiotic resistance. Undergraduate Biology Honors thesis.

Students and Postdoctoral fellows involved:

Post-doc 1. Dr. DeEtta Mills

Graduate Students 2. Dr. Suriya Jayawardena
3. Mr. Kok-Fai Kong
4. Dr. Shalaka Indulkar
5. Dr. Abhideep Joshi

Undergraduate students 6. Mr. Alian Aguila (Past)
7. Mr. Sergio Luna