Molecular epidemiology and antibiotic resistance profiles of invasive Haemophilus influenzae from Norway 2017-2021

Abstract

Invasive Haemophilus influenzae (Hi) disease has decreased in countries that included Hi type b (Hib) vaccination in their childhood immunization programs in the 1990s. Non-typeable (NT) and non-b strains are now the leading causes of invasive Hi disease in Europe, with most cases reported in young children and the elderly. Concerningly, no vaccines toward such strains are available and beta-lactam resistance is increasing. We describe the epidemiology of invasive Hi disease reported to the Norwegian Surveillance System for Communicable Diseases (MSIS) (2017-2021, n = 407). Whole-genome sequencing (WGS) was performed on 245 isolates. We investigated the molecular epidemiology (core genome phylogeny) and the presence of antibiotic resistance markers (including chromosomal mutations associated with beta-lactam or quinolone resistance). For isolates characterized with both WGS and phenotypic antibiotic susceptibility testing (AST) (n = 113) we assessed correlation between resistance markers and susceptibility categorization by calculation of sensitivity, specificity, and predictive values. Incidence rates of invasive Hi disease in Norway ranged from 0.7 to 2.3 per 100,000 inhabitants/year (mean 1.5 per 100,000) and declined during the COVID-19 pandemic. The bacterial population consisted of two major phylogenetic groups with subclustering by serotype and multi-locus sequence type (ST). NTHi accounted for 71.8% (176). The distribution of STs was in line with previous European reports. We identified 13 clusters, including four encapsulated and three previously described international NTHi clones with bla _TEM-1 (ST103) or altered PBP3 (rPBP3) (ST14/IIA and ST367/IIA). Resistance markers were detected in 25.3% (62/245) of the isolates, with bla _TEM-1 (31, 50.0%) and rPBP3 (28, 45.2%) being the most frequent. All isolates categorized as resistant to aminopenicillins, tetracycline or chloramphenicol possessed relevant resistance markers, and the absence of relevant substitutions in PBP3 and GyrA/ParC predicted susceptibility to cefotaxime, ceftriaxone, meropenem and quinolones. Among the 132 WGS-only isolates, one isolate had PBP3 substitutions associated with resistance to third-generation cephalosporins, and one isolate had GyrA/ParC alterations associated with quinolone resistance. The detection of international virulent and resistant NTHi clones underlines the need for a global molecular surveillance system. WGS is a useful supplement to AST and should be performed on all invasive isolates.

Keywords: Haemophilus influenzae (Hi); Norway; antibiotic resistance; epidemiology; invasive; surveillance; vaccine; whole-genome sequencing (WGS).

FIGURE 1

Flow chart showing the data used in the study of invasive Haemophilus influenzae in Norway 2017–2021. Of 407 cases reported to the Surveillance System for Communicable Disease in Norway (MSIS), we identified 268 samples submitted to the national reference laboratory at the Norwegian Institute of Public Health (NIPH) that were whole-genome sequenced (WGS). Of these, 245 isolates were included in the study. Data on antibiotic susceptibility testing (AST) was only available for 113 of the 245 isolates.

FIGURE 2

Number of cases of invasive Haemophilus influenzae disease reported to the Norwegian Surveillance System for Communicable diseases (MSIS) at the Norwegian Institute of Public Health (NIPH) by month in Norway 2017–2019 (blue shaded area, N = 407), compared to the number of isolates whole-genome sequenced at the national reference laboratory at NIPH included in this study (dark blue line, N = 245) and the number of isolates for which antimicrobial testing was performed (blue bars, N = 113).

FIGURE 3

Number of Hi serotypes per year in 245 invasive Haemophilus influenzae isolates from Norway 2017–2021.

FIGURE 4

(A) Unrooted approximately-maximum-likelihood phylogenetic tree based on the core genome (core genes: 1158) of 245 invasive Haemophilus influenzae isolates from Norway 2017–2021. The isolates cluster in two main groups (division I and II, gray shading) and by serotype (leaf node color). (B) Midpoint-rooted approximately-maximum-likelihood phylogenetic tree based on core genome (core genes: 1158) annotated with division, serotype (leaf node color), ST, ftsI allele, markers of antibiotic resistance (genes and mutations), sex, age, sample year and county. The red arrows show clusters containing beta-lactam resistance markers (clusters 2, 8, and 11; Table 1).

FIGURE 5

(A) Midpoint-rooted approximately-maximum-likelihood phylogenetic tree based on core genomes (core genes: 840) of 245 invasive Haemophilus influenzae isolates from Norway 2017–2021 (colored leaf nodes) and 797 publicly available sequences worldwide, annotated with serotypes, STs and transferable resistance genes. The red arrow shows the bla_TEM–1-carrying ST103 NTHi clone. (B) Detailed presentation of the bla_TEM–1-carrying ST103 NTHi clone [red arrow in panel (A)].