Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

Abstract

Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae.

FIG 1

Comparison of H. haemolyticus versus H. influenzae evolutionary lineages. H. haemolyticus and H. influenzae strains analyzed here are labeled with their species abbreviations and the strain names shown in Table 1. (A and B) Phylogenetic trees based on 16S rRNA gene (A) and concatenated MLST loci (B) showing the evolutionary relationships of the H. haemolyticus and H. influenzae genome sequences analyzed here. Percent bootstrap values indicate support for internal nodes on the trees, and the branch length scale bars show P distances. (C) Results of whole-genome sequence comparisons among H. haemolyticus and H. influenzae based on ANI analysis. ANI values (percentages) between pairs of genomes are color coded as shown in the key, and the relationships among the genomes based on these values are shown as dendrograms on both axes.

FIG 2

Homologous gene cluster presence/absence matrix. Genomes are shown as rows, and homologous gene clusters are shown as columns. The presence of a gene cluster in a genome is indicated by green, and cluster absence is indicated by blue. H. haemolyticus genomes are shown on top of the matrix, and H. influenzae genomes are shown below. Core clusters found in all genomes are shown on the right of the matrix, and clusters exclusive to H. haemolyticus (i.e., potential RT-PCR targets) are shown in the upper left corner of the matrix.

FIG 3

H. haemolyticus-specific potential RT-PCR target genes. The presence (green) and absence (blue) of 3 H. haemolyticus-specific genes—hdg, purT, and sod—are shown for the H. haemolyticus (n = 10) and H. influenzae (n = 20) strains analyzed here. Strain names are as shown in Table 1. The percent identities of H. haemolyticus-specific genes to reference genes from strain M19107 are color coded as shown in the key. The sequences of hdg, purT, and sod of six H. haemolyticus strains (marked with asterisks) were determined by Sanger sequencing. Five H. haemolyticus strains were sequenced by WGS. The M19107 strain was characterized by both methods.

FIG 4

Results of RT-PCR assays for the discrimination of H. haemolyticus versus H. influenzae strains. (A) Four possible results for combined purT and hpd RT-PCR assays are evaluated: purT⁺/hpd, purT/hpd⁺, purT⁺/hpd⁺, and purT/hpd. The numbers of H. haemolyticus (red) and H. influenzae (blue) strains with each combination are shown. (B) Sensitivity and specificity values for discrimination of H. haemolyticus and H. influenzae based on purT or hpd RT-PCR assays alone compared to combined RT-PCR assays for H. haemolyticus (purT⁺/hpd) and H. influenzae (purT/hpd⁺).