Beta-Lactamase in Haemophilus ducreyi: Purification, Characterization, and Mutagenesis

Sammanfattning: Chancroid caused by Haemophilus ducreyi, has been described as a significantly predisposing factor of HIV heterosexual transmission in an endemic region of both diseases. Antibiotic resistances are extensively found in H. ducreyi and many of the resistances are due to the presence of resistance plasmids. A high tendency of drug resistances has commonly been found among isolates derived in Thailand. In the research described in this thesis, plasmids of sixty-three H. ducreyi isolates from Thailand were purified and analyzed. Plasmid DNA was further cloned into Escherichia coli and ampicillin resistant transformants were selected and characterized by plasmid profiles and susceptibility patterns to various b-lactams. A 3.6-kb ampicillin resistance plasmid (pCb) was subsequently purified and characterized. The pCb b-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and Chelating Sepharose Fast Flow loaded with Zn2+. The enzyme belonged to a class A b-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322 (TEM-1). Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino acid sequence. Beta-lactamase TEM-1 (pCb) derivatives; E104K, R164D, D179R, E104K+R164D, E104K+D179R, R164D+D179R, and E104K+R164D+D179R were constructed by site-directed mutagenesis. E104K and D179R showed less of a catalytic effect than R164D. The mutant clones containing the R164D substitution showed a reduction in the minimal inhibitory concentration (MIC) of ampicillin. However, the E104K+R164D and E104K+R164D+D179R mutant clones elevated the MIC of ceftazidime and cefotaxime. Furthermore, the dissociation constant for the inhibitor binding value (Ki) of clavulanic acid for the mutant b-lactamases carrying the R164D substitution was much higher than the parental enzyme. In addition, two b-lactamases, penicillinase type I from Bacillus cereus and TEM-1 (pCb) b-lactamase, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni2+ in an active form. Flow-injection analysis of b-lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins.