Phylogenetic Structure and Comparative Genomics of Multi-National Invasive Haemophilus influenzae Serotype a Isolates

Abstract

Recent reports have indicated a rise of invasive disease caused by Haemophilus influenzae serotype a (Hia) in North America and some European countries. The whole-genome sequences for a total of 410 invasive Hia isolates were obtained from 12 countries spanning the years of 1998 to 2019 and underwent phylogenetic and comparative genomic analysis in order to characterize the major strains causing disease and the genetic variation present among factors contributing to virulence and antimicrobial resistance. Among 410 isolate sequences received, 408 passed our quality control and underwent genomic analysis. Phylogenetic analysis revealed that the Hia isolates formed four genetically distinct clades: clade 1 (n = 336), clade 2 (n = 13), clade 3 (n = 3) and clade 4 (n = 56). A low diversity subclade 1.1 was found in clade 1 and contained almost exclusively North American isolates. The predominant sequence types in the Hia collection were ST-56 (n = 125), ST-23 (n = 98) and ST-576 (n = 51), which belonged to clade 1, and ST-62 (n = 54), which belonged to clade 4. Clades 1 and 4 contained predominantly North American isolates, and clades 2 and 3 predominantly contained European isolates. Evidence of the presence of capsule duplication was detected in clade 1 and 2 isolates. Seven of the virulence genes involved in endotoxin biosynthesis were absent from all Hia isolates. In general, the presence of known factors contributing to β-lactam antibiotic resistance was low among Hia isolates. Further tests for virulence and antibiotic susceptibility would be required to determine the impact of these variations among the isolates.

Keywords: Haemophilus influenzae; genomics; invasive isolates; phylogenetic analysis; serotype a.

FIGURE 1

(A) Phylogeny showing the phylogenetic structure of the Hia isolates. Each clade is marked with the number of isolates and the major sequence types and their counts. The SNP difference between each clade is shown. (B) The phylogeny is shown including all Hia isolates and the additional encapsulated serotypes. The four Hia clades are shown in bold.

FIGURE 2

The phylogeny for clade 1 is shown in this figure. Metadata such as region, sequence type and years are included for reference. Other sequence type refers to STs with less than 15 isolates. A red “T” is included next to subclades that contain isolates from both North America and Europe. Subclade 1.1 is labeled in the figure.

FIGURE 3

The capsule regions for Hia isolates belonging to each of the four clades is shown. Clades 1 and 2 contain genes encoding an integrase and transposase proteins and are flanked by IS1016 elements.

FIGURE 4

Sequence alignment of the iga gene consisting of regions 1,100–1,180 with respect to the reference sequence. The three sites of indels are boxed in black, yellow and red. Each variant is labeled with the count of isolates harboring that variant.

FIGURE 5

The PBP3 variants are shown in the table for each of the major Hia clades. The phylogeny consists of PBP3 protein sequences and is annotated with the respective country of the isolate. The branches include which of the four major Hia clades the isolates belong to.