dPCR detecting the protein D (hpd) and fuculose kinase (fucK) genes showed high sensitivity and specificity for identifying Haemophilus influenzae and differentiating it from H. haemolyticus. Phylogenetic analysis using the 16S rRNA gene demonstrated two distinct groups for H. influenzae and H. haemolyticus.
Haemophilus species account for approximately 10% of the bacterial flora in the human respiratory tract (9). At least 8 different Haemophilus species are found to colonize the pharyngeal cavity of humans (13) Discrimination between the two organisms can be challenging due to similarities in colony and cell morphology, biochemical characteristics, and genetic background. Hemolytic H. haemolyticus can be differentiated from H. influenzae by production of clear (beta) hemolysis on horse blood agar. The hemolysis activity may be lost after subculture, however, and nonhemolytic H. haemolyticus strains are often misidentified as H. influenzae (10,13,15,16,18).DNA-DNA hybridization is the gold standard for identifying bacterial species (2-4). Because of the complexity of DNA-DNA hybridization, 16S rRNA gene sequence similarity is widely used as a tool to identify bacteria at the species level and assist with differentiating between closely related bacterial species (8). Several molecular tools have been assessed for differentiation of H. haemolyticus from H. influenzae, such as the fuculose kinase gene fucK, the H. influenzae adherence and penetration protein gene hap, the [Cu, Zn]-superoxide dismutase gene sodC, the immunoglobulin A protease gene iga, the lipopolysaccharide (LPS) gene lgtC, and the outer membrane protein P6 (1, 16-18, 24, 26). fucK has been proposed as a reliable marker gene for discrimination of H. influenzae from other Haemophilus species (17). A real-time PCR assay targeting the Haemophilus protein D gene (hpd) has been developed to identify H. influenzae (28). The hpd assay is highly specific for H. influenzae and provides a potential tool to differentiate H. influenzae from H. haemolyticus, including nonhemolytic H. haemolyticus. In this study, both hpd and fucK PCR assays were evaluated against 16S rRNA gene phylogeny for distinguishing H. influenzae from H. haemolyticus by the use of Haemophilus isolates identified during a carriage study in Minnesota (12,22 All isolates were further characterized using the hpd and fucK PCR assays with crude DNA as the template (28). All of the 31 hemolytic H. haemolyticus and 128 other Haemophilus species isolates were negative by both hpd and fucK PCR assays. Of the 245 presumed H. influenzae isolates, 121 (49.4%) were positive for both the hpd and fucK genes, which is the expected genotype for H. influenzae, and 98 (40.0%) were negative for both hpd and fucK,