Classification, identification, and clinical significance of Haemophilus and Aggregatibacter species with host specificity for humans

Abstract

The aim of this review is to provide a comprehensive update on the current classification and identification of Haemophilus and Aggregatibacter species with exclusive or predominant host specificity for humans. Haemophilus influenzae and some of the other Haemophilus species are commonly encountered in the clinical microbiology laboratory and demonstrate a wide range of pathogenicity, from life-threatening invasive disease to respiratory infections to a nonpathogenic, commensal lifestyle. New species of Haemophilus have been described (Haemophilus pittmaniae and Haemophilus sputorum), and the new genus Aggregatibacter was created to accommodate some former Haemophilus and Actinobacillus species (Aggregatibacter aphrophilus, Aggregatibacter segnis, and Aggregatibacter actinomycetemcomitans). Aggregatibacter species are now a dominant etiology of infective endocarditis caused by fastidious organisms (HACEK endocarditis), and A. aphrophilus has emerged as an important cause of brain abscesses. Correct identification of Haemophilus and Aggregatibacter species based on phenotypic characterization can be challenging. It has become clear that 15 to 20% of presumptive H. influenzae isolates from the respiratory tracts of healthy individuals do not belong to this species but represent nonhemolytic variants of Haemophilus haemolyticus. Due to the limited pathogenicity of H. haemolyticus, the proportion of misidentified strains may be lower in clinical samples, but even among invasive strains, a misidentification rate of 0.5 to 2% can be found. Several methods have been investigated for differentiation of H. influenzae from its less pathogenic relatives, but a simple method for reliable discrimination is not available. With the implementation of identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry, the more rarely encountered species of Haemophilus and Aggregatibacter will increasingly be identified in clinical microbiology practice. However, identification of some strains will still be problematic, necessitating DNA sequencing of multiple housekeeping gene fragments or full-length 16S rRNA genes.

FIG 1

Genetic relationships of Haemophilus and Aggregatibacter species, using Escherichia coli as an outgroup. Concatenated sequences of near-full-length 16S rRNA genes (1,361 to 1,364 nt) plus fragments of three housekeeping genes, infB, pgi, and recA (1,293 nt), were compared by the neighbor-joining method (36, 99). The dendrogram is based on type strains of validated species, except for H. ducreyi, where strain 35000HP was used (GenBank accession no. AE017143). Strain CCUG 11096 represents the not validly named species H. intermedius. Bar, 2 substitutions per 100 nucleotides.

FIG 2

NAD utilization in Pasteurellaceae. Nicotinamide mononucleotide (NMN) and NAD enter the periplasm through the general porin OMP P2 and are degraded to nicotinamide riboside (NR); exogenous NR probably enters the periplasm through a different porin (293). NR is internalized through a cytosolic membrane-located permease (PnuC) and serves as the substrate for a resynthesizing enzyme which uses ATP to generate NAD. Nicotinamide (Nam) freely diffuses through the cell membranes and can serve as the substrate for those members of the family that express a functional nicotinamide phosphoribosyltransferase (NadV). (Based on reference .)

FIG 3

Neighbor-joining dendrogram based on concatenated gene fragments of adk, atpG, frdB, mdh, pgi, and recA (2,712 nucleotides), comparing the type strains of H. influenzae, H. aegyptius, and H. haemolyticus (filled circles), five genome-sequenced H. haemolyticus strains (294), and 30 strains of H. haemolyticus and related organisms (36), with 900 H. influenzae sequence types downloaded from the MLST website (www.mlst.net). The 36 strains of H. haemolyticus and related organisms are negative for fucK, and gaps were treated by complete deletion using MEGA, version 5 (295). Phylogenetic group I is indicated in blue, phylogenetic group II in yellow, and H. haemolyticus and related organisms in red. Strains with equivocal allocation to phylogenetic groups are shown in gray. Bar, 1 substitution per 200 nucleotides.

FIG 4

The type strain of H. pittmaniae was cultured for 24 h on different agars in the presence of a NAD-containing disk and photographed by transillumination. (A) Todd-Hewitt agar. Growth is apparent only around the NAD disk. (B) Five percent horse blood agar. Growth of hemolytic colonies is visible over the entire agar. (Reprinted from reference with permission of the publisher.)

FIG 5

Neighbor-joining tree based on near-full-length 16S rRNA gene sequences (1,361 or 1,362 nucleotides), comparing the type strains of H. influenzae, H. aegyptius, and H. haemolyticus (filled circles) with 80 strains of H. influenzae (36, 199, 296) and 39 reference strains of H. haemolyticus and related organisms (36, 195, 199, 294) (filled triangles). Strain PN134 was omitted from the comparison because of doubtful identification to the species level (199). Phylogenetic group I is indicated in blue, phylogenetic group II in yellow, and H. haemolyticus and related organisms in red. H. haemolyticus and related organisms are located on two branches, with one adjacent to H. influenzae phylogenetic group II and composed mainly of non-hemolytic H. intermedius subsp. gazogenes (bootstrap support, 36%) and a larger cluster encompassing the type strain, porphyrin-synthesizing strains, and cryptic genospecies biotype IV strains (bootstrap support, 57%). In contrast, all representatives of H. influenzae and the type strain of H. aegyptius are located in a single cluster supported by a bootstrap value of 63%. Analysis was conducted using MEGA5 (295), and ambiguous positions were removed for each sequence pair.

FIG 6

The three 16S rRNA gene helix 18 types present in a collection of isolates from the H. influenzae group, represented by H. influenzae strain Rd, cryptic genospecies biotype IV strain 16N, and H. haemolyticus strain NCTC 10659^T. The outer stem of helix 18 (nt 453 to 477) is shown. Conserved nucleotides are shown in blue. (Adapted from reference with permission of the publisher.)