First Whole-Genome Sequence of a Haemophilus influenzae Type e Strain Isolated from a Patient with Invasive Disease in Italy.

In the present era of conjugate vaccines against Haemophilus influenzae type b, non-vaccine-preventable strains are of concern. Here, we report the first whole-genome sequence of an invasive H. influenzae type e strain. This genomic information will enable further investigations on encapsulated non-type b H. influenzae strains.

GENOME ANNOUNCEMENT

Haemophilus influenzae is a Gram-negative bacterium capable of causing respiratory tract infections as well as severe invasive disease. This species is characterized by the presence/absence of a polysaccharide capsule (1). Encapsulated H. influenzae expresses one of the six structurally and antigenically distinct capsular polysaccharides (serotypes a to f); nonencapsulated isolates are commonly referred to as nontypeable H. influenzae (NTHi). Since the introduction of conjugate vaccines against H. influenzae type b (Hib), invasive disease caused by this serotype has drastically decreased (2). However, invasive disease due to H. influenzae has not been eliminated, and NTHi and encapsulated non-type b H. influenzae strains are nowadays responsible for most invasive infections (3, 4). In Italy, NTHi was found to predominate among invasive isolates, but a number of cases were associated with type e and type f strains (5). In particular, invasive H. influenzae type e (Hie) disease has been observed since the beginning of the 2000s in our country and, recently, Hie strains were found to account for the 9% of all invasive H. influenzae isolates (5–7). In this study, we determined the whole-genome sequence of an Hie strain isolated from the cerebrospinal fluid from a 66-year-old patient with meningitis in 2014, in Italy. The H. influenzae isolate was sent to the reference laboratory of the National Surveillance of Invasive Bacterial Disease at the Istituto Superiore di Sanità (Rome, Italy), where, following genotyping confirmation of the serotype (8), we subjected it to whole-genome sequencing. To our knowledge, no published whole-genomic data are available for Hie.

Genomic DNA was extracted from an overnight culture at 37°C using the NucleoSpin DNA extract kit (Macherey-Nagel, Duren, Germany). Whole-genome sequencing was performed using Illumina MiSeq (250-bp paired-end reads) technology (Illumina, San Diego, CA). The genome sequences were assembled de novo using Newbler (9). The final assembly consists of 1,850,604 bp in 140 contigs, with an N50 of 29,763 bp. The resulting coverage was 310×. Genome annotation was performed using Glimmer3 (10) and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/), and 1,873 putative protein-coding genes were identified. No plasmid or resistance gene was found by submitting the genome at the Center for Genomic Epidemiology (CGE) server (https://cge.cbs.dtu.dk/services/), using PlasmidFinder and ResFinder, respectively. Multilocus sequence type (MLST) was assigned according to the H. influenzae MLST website scheme (http://pubmlst.org/hinfluenzae/) and it was sequence type 18 (ST18). Various putative genes encoding virulence factors were identified, including adherence factors (hap and type IV pili), endotoxins (kdsB and lpxK), lipooligosaccharide (LOS) (galE and lic), and host immune evasion proteins (mrsA and iga1).

Detailed analysis of the reported genome, including comparisons with other encapsulated and nonencapsulated H. influenzae genomes, is under way. The whole-genome sequence reported here will provide a reference data set for Hie, improving our understanding on the genomic diversity of encapsulated non-type b H. influenzae strains.

Accession number(s).

This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. MTBH00000000. The version described in this paper is version MTBH01000000.