New insights into the mechanisms of ß-Lactam resistance in Haemophilus influenzae.

Despite the benefit of vaccination against Haemophilus influenzae type b in developed countries, respiratory tract infections with non encapsulated strains remain a significant problem that is compounded by increasing antibiotic resistance in H. influenzae, particularly to ß-lactam antibiotics. The most common mechanism of resistance to ß-lactam antibiotics in H. influenzae is the production of a ß-lactamase, usually TEM-1 or occasionally ROB-1. Less commonly, the resistance is associated with altered penicillin binding protein 3 (PBP3) to produce ßlactamase negative ampicillin resistant (BLNAR) strains. Single nucleotide specific PCR and multiplex PCR assays were developed to differentiate the ß-lactamase genes of H. influenzae. These assays were able to differentiate ROB-1 from TEM-1 producers, and further differentiate the promoter type of the blaTEM genes into Pa/Pb, Pdel and Prpt, with the latter two representing promoter types discovered during the study. In the study, 89 TEM-1 ß-lactamase positive clinical isolates of H. influenzae were examined, and the relative percentages of the promoter types were 60, 20 and 20% for Pdel, Prpt and Pa/Pb respectively. Significantly, the P3 promoter which is the most common type associated with blaTEM-1 in Enterobacteriaceae, was not found. Cloning experiments demonstrated that while TEM type extended spectrum ß-lactamases (ESBLs) and inhibitor resistant TEM ß-lactamases (IRTs) would significantly raise the cefotaxime and amoxicillin-clavulante MICs respectively in recombinant strains, the MICs would not be high enough to exceed the CLSI resistance breakpoints. Expression of these ß-lactamase genes in strains with altered PBP3 further increased the levels of resistance. A disc diffusion screening test was developed to screen for ESBLs in H. influenzae, and was able to differentiate recombinant strains producing ESBLs from other strains with naturally occurring resistance mechanisms. Two cefotaxime and amoxicillin-clavulanate resistant clinical isolates of H. parainfluenzae were extensively characterised. Both strains gave a positive ESBL disc diffusion screening test, and were found to produce both a TEM-15 ESBL and a TEM-1 ß-lactamase. The strains had identical 3.7kb plasmids bearing a blaTEM-15 gene transcribed from a Prpt promoter. Both strains also had a chromosomally integrated blaTEM-1, and in one of the strains was transcribed from a novel promoter (Pdel17) produced from a 17bp deletion of the Pa/Pb promoter. Both strains also had deduced amino acid substitutions in PBP3 similar to those found in BLNAR strains of H. influenzae. Studies involving transformation of various control strains with the blaTEM-15 indicate that the combination of blaTEM-15 and the altered PBP3 was responsible for the very high cefotaxime MICs. This was the first report of an ESBL in a Haemophilus spp., and the first report of altered PBP3 in a Haemophilus sp. other than H. influenzae. Characterisation of a limited collection of ROB-1 producing strains has shown that the location of the blaROB-1 gene on the small mobilisable plasmid pB1000 appears to be widespread. The inefficiency with which this plasmid is mobilised might account for the relatively low frequency with which ROB-1 positive strains are encountered. Variation in cefaclor MIC is not associated with variation in blaROB-1 sequence, but may be associated with a previously unrecognised heterogeneity in the PBP3 background of the host strains. In summary, the major findings of this work are: 1) An insight into how various possible genotypic changes in antibiotic resistance genes in H. influenzae would affect the resistance phenotype. 2) The prediction of the emergence of a new resistance mechanism and subsequent characterization of the strains in which this was found. 3) Data, screening tests and guidelines that will improve both the detection of antibiotic resistance in H. influenzae and the interpretation of susceptibility testing result