Genomic characterization of Haemophilus influenzae: a focus on the capsule locus

Abstract

Background: Haemophilus influenzae (Hi) can cause invasive diseases such as meningitis, pneumonia, or sepsis. Typeable Hi includes six serotypes (a through f), each expressing a unique capsular polysaccharide. The capsule, encoded by the genes within the capsule locus, is a major virulence factor of typeable Hi. Non-typeable (NTHi) does not express capsule and is associated with invasive and non-invasive diseases.

Methods: A total of 395 typeable and 293 NTHi isolates were characterized by whole genome sequencing (WGS). Phylogenetic analysis and multilocus sequence typing were used to characterize the overall genetic diversity. Pair-wise comparisons were used to evaluate the capsule loci. A WGS serotyping method was developed to predict the Hi serotype. WGS serotyping results were compared to slide agglutination (SAST) or real-time PCR (rt-PCR) serotyping.

Results: Isolates of each Hi serotype clustered into one or two subclades, with each subclade being associated with a distinct sequence type (ST). NTHi isolates were genetically diverse, with seven subclades and 125 STs being detected. Regions I and III of the capsule locus were conserved among the six serotypes (≥82% nucleotide identity). In contrast, genes in Region II were less conserved, with only six gene pairs from all serotypes showing ≥56% nucleotide identity. The WGS serotyping method was 99.9% concordant with SAST and 100% concordant with rt-PCR in determining the Hi serotype.

Conclusions: Genomic analysis revealed a higher degree of genetic diversity among NTHi compared to typeable Hi. The WGS serotyping method accurately predicted the Hi capsule type and can serve as an alternative method for Hi serotyping.

Keywords: Capsule locus; Genetic diversity; Haemophilus influenzae; Multilocus sequence typing; Serotype; Whole genome sequencing.

Fig. 1

Population structure of H. influenzae isolates. The genomic relatedness of 688 Hi isolates is depicted as a maximum likelihood phylogeny. The three main clades (I, II, and III) are labeled on the tree and each isolate is color coded by the serotype, as determined by slide agglutination. The NTHi isolates within the serotype-specific subclades are denoted by ψ, σ, or *. The ψ indicates the isolate that had discrepant serotyping results between WGS and SAST methods. The σ indicates the only two NTHi isolates that contained an internal stop codon within a capsule gene. The * indicates the remaining five NTHi isolates (capsule nulls) that were detected within serotype-specific subclades. The tree scale is 0.1 substitutions per site along the length specified. Bootstrap values were 100/100 for all major nodes

Fig. 2

Genetic organization of the H. influenzae capsule genes. All serotypes contained the Region I genes bexABCD (black) and the Region III genes (gray): hcsA and hcsB. The Region II genes were divergent among serotypes. Each arrow represents a different gene and is labeled with the gene name. The Region II arrows are color coded by serotype

Fig. 3

The allelic variation detected for each Region I, II and III gene. Each capsule gene from Regions I and III (a) or Region II (b) are listed on the y-axis. The unique number of alleles detected per gene is denoted within the parentheses. The number of genomes associated with that serotype is also provided. The x-axis depicts the percent identity shared among the nucleotide alleles for each capsule gene. The left side of the bar represents the minimum sequence identity and the right side represents the maximum sequence identity detected. If only one allele was detected, no data are shown

Fig. 4

WGS serotyping method for determining H. influenzae serotype from WGS data. The three main steps of this process include identifying the capsule genes, predicting the capsule gene expression, and assigning the predicted serotype