RESUMO
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.
RESUMO
Non-typeable Haemophilus influenzae (NTHi), a commensal organism in pre-school children, is an opportunistic pathogen causing respiratory tract infections including acute otitis media. Adults suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized by NTHi. Previous research has suggested that, in some bacterial species, the intracellular elongation factor thermo-unstable (EF-Tu) can moonlight as a surface protein upon host encounter. The aim of this study was to determine whether EF-Tu localizes to the surface of H. influenzae, and if such surface-associated EF-Tu is a target for bactericidal antibodies. Using flow cytometry, transmission immunoelectron microscopy, and epitope mapping, we demonstrated that EF-Tu is exposed at the surface of NTHi, and identified immunodominant epitopes of this protein. Rabbits immunized with whole-cell NTHi produced significantly more immunoglobulin G (IgG) directed against EF-Tu than against the NTHi outer membrane proteins D and F as revealed by enzyme-linked immunosorbent assays. Chemical cleavage of NTHi EF-Tu by cyanogen bromide (CNBr) followed by immunoblotting showed that the immunodominant epitopes were located within the central and C-terminal regions of the protein. Peptide epitope mapping by dot blot analysis further revealed four different immunodominant peptide sequences; EF-Tu41-65, EF-Tu161-185, EF-Tu221-245, and EF-Tu281-305. These epitopes were confirmed to be surface-exposed and accessible by peptide-specific antibodies in flow cytometry. We also analyzed whether antibodies raised against NTHi EF-Tu cross-react with other respiratory tract pathogens. Anti-EF-Tu IgG significantly detected EF-Tu on unencapsulated bacteria, including the Gram-negative H. parainfluenzae, H. haemolyticus, Moraxella catarrhalis and various Gram-positive Streptococci of the oral microbiome. In contrast, considerably less EF-Tu was observed at the surface of encapsulated bacteria including H. influenzae serotype b (Hib) and Streptococcus pneumoniae (e.g., serotype 3 and 4). Removal of the capsule, as exemplified by Hib RM804, resulted in increased EF-Tu surface density. Finally, anti-NTHi EF-Tu IgG promoted complement-dependent bacterial killing of NTHi and other unencapsulated Gram-negative bacteria as well as opsonophagocytosis of Gram-positive bacteria. In conclusion, our data demonstrate that NTHi EF-Tu is surface-exposed and recognized by antibodies mediating host innate immunity against NTHi in addition to other unencapsulated respiratory tract bacteria.