Genetic and molecular characterization of beta-lactamase-negative ampicillin-resistant Haemophilus influenzae with unusually high resistance to ampicillin

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.

FIG. 1.

Affinity of recombinant PBP3 proteins for fluorescent-labeled Bocillin. One hundred-seventy nanograms of each PBP3 was incubated for 30 min at 37°C with 2 to 12 μM Bocillin. Error bars indicate ±1 standard deviation. PBP3 proteins tested were expressed from ftsI of H. influenzae Rd (control); BLNAR strains 1312 (group I), 1311, and 1369 (group II); and single-substitution alleles of group I (Arg517 to His) and group II (Asn526 to Lys) PBP3.

FIG. 2.

Transpeptidase activity of each recombinant PBP3 expressed in relative fluorescent units. Reaction mixtures contain 2.4 μg of each PBP3. Error bars represent means ± 1 standard deviation.

FIG. 3.

Accumulation of [¹⁴C]erythromycin in strains of H. influenzae. Cells were grown and treated as described by Sánchez et al. (32). The experiment was started after 5 min of preincubation of the cells by the addition of radiolabeled erythromycin. After 5 min, CCCP was added to a final concentration of 0.2 mM. Open symbols represent untreated cultures; closed symbols represent CCCP-treated cultures. Each datum point represents the average of two experiments. CPM, counts per minute.