Whole-Genome Sequencing of Aggregatibacter Species Isolated from Human Clinical Specimens and Description of Aggregatibacter kilianii sp. nov.

Aggregatibacter species are commensal bacteria of human mucosal surfaces that are sometimes involved in serious invasive infections. During the investigation of strains cultured from various clinical specimens, we encountered a coherent group of 10 isolates that could not be allocated to any validly named species by phenotype, mass spectrometry, or partial 16S rRNA gene sequencing. Whole-genome sequencing revealed a phylogenetic cluster related to but separate from Aggregatibacter aphrophilus The mean in silico DNA hybridization value for strains of the new cluster versus A. aphrophilus was 56% (range, 53.7 to 58.0%), whereas the average nucleotide identity was 94.4% (range, 93.9 to 94.8%). The new cluster exhibited aggregative properties typical of the genus Aggregatibacter Key phenotypic tests for discrimination of the new cluster from validly named Aggregatibacter species are alanine-phenylalanine-proline arylamidase, N-acetylglucosamine, and ß-galactosidase. The name Aggregatibacter kilianii is proposed, with PN_528 (CCUG 70536T or DSM 105094T) as the type strain.

Substitutions in PBP3 confer resistance to both ampicillin and extended-spectrum cephalosporins in Haemophilus parainfluenzae as revealed by site-directed mutagenesis and gene recombinants.

Objectives: To determine the association of amino acid substitutions in PBP3 with ß-lactam susceptibility in Haemophilus parainfluenzae. Methods: Single and multiple amino acid mutations at positions 385, 511 and 526 were introduced into PBP3 of a ß-lactam-susceptible H. parainfluenzae strain using site-directed mutagenesis. Recombinants were also generated using PCR-amplified ftsI from clinical strains encoding multiple amino acid substitutions. MICs of ampicillin, cefuroxime, cefotaxime and ceftriaxone were determined using Etest®. Results: Transformation of a susceptible strain with ftsI from clinical strains encoding four substitutions in the transpeptidase region of PBP3 conferred resistance to ampicillin, but not to cephalosporins. Introduction of ftsI from a clinical strain encoding eight substitutions conferred resistance to ampicillin, cefotaxime and ceftriaxone. MICs for recombinants were lower than those for the donor strains. Using site-directed mutagenesis, no single substitution conferred resistance to the tested ß-lactams, although V511A increased the MIC of cefuroxime to the intermediate category for intravenous administration. Recombinants encoding N526K/H/S in combination with V511A were resistant to ampicillin. Substitution S385T increased the MICs of third-generation cephalosporins if V511A was also present. Conclusions: Substitutions in PBP3 are sufficient to confer resistance to both ampicillin and third-generation cephalosporins in H. parainfluenzae. A combination of substitutions at positions Val-511 and Asn-526 confers resistance to ampicillin. Resistance to third-generation cephalosporins probably requires more than four substitutions in PBP3.