Abstract
Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 micro g/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 micro g/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously (K. Ubukata, Y. Shibasaki, K. Yamamoto, N. Chiba, K. Hasegawa, Y. Takeuchi, K. Sunakawa, M. Inoue, and M. Konno, Antimicrob. Agents Chemother. 45:1693-1699, 2001). Geometric mean ampicillin MICs for several clinical isolates were 8 to 10.56 micro g/ml. Replacement of the ftsI gene in H. influenzae Rd with the intact ftsI from several clinical isolates resulted in integrants with typical BLNAR geometric mean ampicillin MICs of 1.7 to 2.2 micro g/ml. Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a, -1b, and -2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 micro g/ml) lowered the ampicillin MIC to 3.67 micro g/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.
Highlights
Despite the extensive utilization of antimicrobial therapies and the availability of the Haemophilus influenzae type b vaccine, H. influenzae remains a major pathogen in bronchopulmonary as well as ear, nose, and throat infections
Clairoux et al [6] studied BLNAR H. influenzae isolated in Canada and found that lowlevel resistance to ampicillin correlated with decreased affinity of 125I-labeled penicillin for PBP3A and -3B in clinical isolates compared to that of H. influenzae Rd
Twenty-six H. influenzae BLNAR isolates were obtained from the Clinical Microbiology Insti
Summary
Despite the extensive utilization of antimicrobial therapies and the availability of the Haemophilus influenzae type b vaccine, H. influenzae remains a major pathogen in bronchopulmonary as well as ear, nose, and throat infections. Clairoux et al [6] studied BLNAR H. influenzae isolated in Canada and found that lowlevel resistance to ampicillin correlated with decreased affinity of 125I-labeled penicillin for PBP3A and -3B in clinical isolates compared to that of H. influenzae Rd. Ampicillin MICs for these BLNAR strains clustered into three groups: group 1 was 0.5 to 1.0 g/ml, group II was 2 to 4 g/ml, and group III had higher MICs of Ն8 g/ml. In group III strains, three residues (Met-377, Ser385, and Leu-389) were replaced by Ile, Thr, and Phe, respectively, in addition to replacement of Asn-526 with Lys. Transformation of H. influenzae Rd with the ftsI gene of the clinical strains produced transformants with ampicillin and cephalosporin MICs equal to those for the original BLNAR strains (1 to 4 g/ml). Some strains had a 7-bp deletion in dacB, which encodes PBP4, this change did not appear to correlate with elevated ampicillin MICs [35]
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