GENETIC-CHARACTERIZATION OF TRIMETHOPRIM RESISTANCE IN HAEMOPHILUS-INFLUENZAE

We previously demonstrated that trimethoprim (Tmp) resistance in Haemo philus influenzae is mediated by chromosomally encoded dihydrofolate r eductase (DHFR),vith a modified primary structure and distinct kinetic properties, To gain insight into the relationship of the DHFR structu re and the level of Tmp resistance that it confers on the host bacteri um, we cloned and characterized the folH genes of one Tmp-susceptible and two Tmp-resistant H. influenzae strains, Differences were observed between Tmp-susceptible and Tmp-resistant isolates both in the promot er region and in the coding sequences, The effect of differences betwe en H. influenzae folH genes on Tmp susceptibility was investigated in Escherichia coli, Various folH gene hybrids were constructed, and thei r influence on Tmp susceptibility was determined, Resistance in E. col i mediated by folH from N. influenzae strain R1047 was associated,vith alterations in the promoter and the central part of folH. In contrast , the E. coli Tmp resistance phenotype associated with the folH gene o f H. influenzae R1042 was characterized by alterations in one or more of three amino acid residues at the C-terminal part of the protein, Th ese data indicate that Tmp resistance is not only related to alteratio ns in the promoter region of the folH gene and the Tmp binding domains at the N-terminal and central part of DHFR, Alterations in the C-term inal part may also cause Tmp resistance, probably as a result of a cha nge in secondary structure and the subsequent loss of Tmp binding affi nity.