The Bacteroides are one of the least studied but most important groups of bacteria to the health of humans. These obligate anaerobic bacteria are the predominant indigenous species (30% of total) in the intestinal tract where they are important to the host's normal physiological functions. However, they are also significant opportunistic pathogens whose infections often require surgical and antimicrobial treatment. Due to inherent and acquired resistance to many antibiotics, Bacteroides infections are becoming increasingly difficult to treat. Thus the long range goals of this research program seek to elucidate the novel mechanisms that regulate expression of antibiotic resistance and virulence determinants in these important organisms. The present proposal specifically addresses resistance to beta-lactam antibiotics and the rapid evolution to high level beta-lactam resistance recently seen in clinical isolates of Bacteroides fragilis. The predominant beta-lactamase (cephalosporinase) producers generally fall into three broad expression classes and it is proposed that these represent stages in the evolution toward high level resistance resulting from alterations in the transcription regulatory regions of the gene(s). The experiments described in this application will examine these alterations and determine the molecular mechanisms that regulate beta-lactamase expression. This will be accomplished by the isolation of beta-lactamase genes from the three expression classes and analysis of their structure at the nucleotide sequence level. Transcription start sites will be determined and the beta-lactamase promoters identified. Using newly developed operon fusion vectors, promoter activity will be analyzed in Bacteroides host strains and compared to other cloned promoters. These comparisons and analyses will allow an accurate assessment of the signals required for initiation of transcription in Bacteroides. Taken together, these data will be used to formulate a model of beta-lactamase expression and document the changes leading to high level expression. Predictions from the model will be tested by the isolation and analysis of mutants with altered expression characteristics. Finally, genes for beta-lactamase from strains resistant to broad spectrum beta-lactams will be characterized as described above. By comparison to the predominant Bacteroides genes and published beta-lactamase sequences, the origins of these novel genes will be determined.
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