In Belgium, the surveillance of H. influenzae infections is based on mandatory reporting of invasive H. influenzae infections (limited to Hib in Flanders and Wallonia). Since 1991, the sentinel laboratories network has also been responsible for reporting all samples from normally sterile sites that have tested positive for H. influenzae by culture or DNA detection (ECDC case definition). Furthermore, data from the National Reference Center (NRC), which has been collecting and analyzing all invasive strains since 2011, contributes to the surveillance efforts. While these surveillance systems enable tracking trends in invasive infections, they do not provide the exact number of such infections and lack information on non-invasive strains and their profiles. [4].
A total of 408 H. influenzae strains were examined in this study, collected from more than 40 clinical laboratories all over the country between November 2021 and April 2022.
The number of strains collected during the national surveillance (n = 352) fell short of expectations. This was due to two factors: first, strains that had to be excluded due to insufficient information or a lack of growth; and second, the challenge of accurately categorizing strains as infection or carriage strains based on the examinations conducted in each laboratory. The number of easily identifiable "infection" strains ultimately outnumbered those of "carriage" strains, which are challenging to isolate from salivary flora. While participation in the surveillance was voluntary, the number of strains collected per province appeared to align with the country's population distribution. The slight overrepresentation of strains from the Flemish region, particularly infection strains in West Flanders, can be attributed to the active participation of Flemish laboratories. Concurrently, the recruitment method, which was aligned with the number of laboratories per province/region and the previously mentioned difficulty in isolating carriage strains, explains the lower recruitment in the Brussels-Capital region, especially for carriage strains.
The number of invasive strains collected during the study period is also notably lower than anticipated (n = 56). In 2022, the NRC diagnosed 146 invasive cases, with 88 in 2021, 84 cases in 2020, and 162 in 2019. This translates to an incidence of 1.3 cases per 100,000 inhabitants in 2022, 0.8 in 2021, 0.7 in 2020, and 1.4 in 2019 [7]. A decline in the incidence of invasive respiratory secretion-transmitted diseases, including H. influenzae infections, was observed in numerous countries, including Belgium, during the COVID-19 pandemic. This decline was attributed to the implementation of confinement and social distancing measures [8]. In Brussels, Flemish Brabant, and Antwerp, the substantial proportion of invasive infections might be correlated with population density. Nevertheless, the limited number of cases and the impact of the COVID-19 crisis on respiratory secretion-transmitted diseases infections in 2021 render the interpretation of the data challenging.
Based on urease production, indole, and ornithine decarboxylase, it is possible to identify a maximum of eight biotypes of H. influenzae. In 1976, Kilian categorized H. influenzae into the initial five biotypes and observed a correlation between specific biotypes and particular disease entities. For instance, almost all biotype I organisms were isolated from patients with acute infections whereas biotype II and III were isolated from normal throats or from patients with conjunctivitis (or various infections for biotype II) [9]. In the current study, biotype II constitutes more than 40% in each studied group, followed by biotypes III and I, accounting for more than 25% and about 15%, respectively. This observation is consistent with a recent investigation focused on H. influenzae carriage in Belgian children attending day care centers or experiencing acute otitis media [10]. In contrast, the frequency of biotype I, presumed to be associated with pathogenicity, did not significantly differ between the invasive, infection, and carriage groups in our study.
The overwhelming majority of studied strains are NTHi, constituting 97.9%, 99.0%, and 85.7% of strains among carriage, infection, and invasive strains, respectively. As in the children study, the number of encapsulated strains is notably higher in the invasive group (14.3% vs 1–2%; p < 0.001). This proportion of NTHI among invasive strains is also consistent with that observed in other European countries, including Spain (85.3%) and Ireland (83%, 95% among non-invasive strains). The primary serotypes implicated in invasive infections are serotype a (7.1%), followed by serotypes f (5.4%) and b (1.8%). No definitive conclusions can be drawn from observations on surveillance strains, given the anecdotal nature of the capsulated cases. This emphasizes the value of monitoring all strains associated with invasive infections, beyond just Hib strains, and underscores the importance of vaccination against other serotypes, such as serotype a, or even the necessity for future pan-serotype H. influenzae vaccines [11].
The susceptibility profile of H. influenzae strains has undergone changes in recent years, marked by the emergence of strains exhibiting reduced susceptibility to beta-lactams in Belgium. Mutations in the ftsI gene not only affect ampicillin susceptibility but can also influence the response to amoxicillin-clavulanic acid combination, as well as second and third-generation cephalosporins, and even meropenem [7]. Therefore, the detection of a beta-lactamase as the sole susceptibility test is no longer sufficient, as it alone does not determine whether the strain is susceptible to ampicillin or not. In the current study, a beta-lactamase enzyme was identified in 18.3%, 19.0%, and 12.5% of carriage, infection, and invasive cases, respectively. Meanwhile, ampicillin resistance was respectively observed in 27.5%, 27.1%, and 19.6% of the cases. These findings are consistent with recent data from various European countries. A Polish study, examining lower respiratory tract infections between 2005 and 2019, reported 74.4% of β-lactamase negative ampicillin-susceptible (BLNAS)[12]. In Spain (2014–2019), invasive strains displayed a 17.6% ampicillin resistance, while in Ireland (2010–2018) it was 18%, in Italy (2017–2021) it was 21.7%, and in Germany (2016–2019) it reached 21.9%, closely resembling our observed rate of 19.6% [13–16]. Additionally, in Norway (2017–2021), 18.6% of invasive strains exhibited ampicillin resistance, with 13.3% attributed to beta-lactamase production (12.5% in our panel) [17]. Resistance to the amoxicillin-clavulanic acid combination, primarily associated with BLNAR featuring multiple substitutions in PBP3, accounted for approximately 7% in the surveillance strains and 10.7% in invasive strains, slightly higher than in Italy (4.9%), Germany (1.7%), or Norway (1.8%).
Modifications in PBP3 may result in variable degrees of reduced susceptibility to beta-lactams, leading to strains with a seemingly susceptible phenotype but MICs approaching the cutoff determined by EUCAST. As a result, our NRC conducts ftsI gene sequencing for any strain displaying diminished susceptibility to beta-lactams. In this study, twenty percent of the analyzed strains exhibited mutations in the ftsI gene, irrespective of the category. Interestingly, among the 352 surveillance strains, three carried mutations in the ftsI gene associated with high-level resistance (group III), a feature not observed in invasive strains. Moreover, these three strains also displayed the S357N substitution linked to cefuroxime resistance, with phenotypic testing confirming a MIC > 256 mg/mL [18]. Four strains belonging to group IIb also showed MICs > 256 for cefuroxime. The identified mutations in their ftsI gene—D350N, M377I, A502V, N526K—constitute the most prevalent combination of amino acid substitutions observed in a previous subset of Belgian β-lactamase-negative ampicillin-resistant strains (BLNAR), and are also found in Czech Republic strains at a rate of 35% [19]. Additionally, five out of the seven cefuroxime-resistant strains, evenly distributed across the three groups, exhibited resistance to cefotaxime. The overall resistance to third-generation cephalosporins (C3G) at 1.2% aligns with rates found in other European countries. In four strains, a decreased susceptibility to beta-lactams was observed, with no amino acid substitutions in the transpeptidase region of PBP3, suggesting a different resistance mechanism. While multidrug-resistant (MDR) H. influenzae has been previously described, the mechanisms underlying the emergence of betalactams resistant isolates are not fully understood. Spontaneous mutations are considered a primary cause of substitutions, and the process of horizontal gene transfer through classical transformation, such as horizontal gene transfer of blaTEM-1 and recombination could also play a role in the further development and dissemination of resistance [20–21].
Michel et al. studied the interspecies recombination with H. parainfluenzae and H. influenzae within their ftsI and mur-E-mur-F PBP-encoding genes and the rearrangement occurence in ompP2 [22]. They suggest that the point mutations in different genes related to AMR and the increasing of intra- and inter-species recombination events is a possible source of progressive loss of susceptibility to antibiotics in chronic carriage.
While resistance to ciprofloxacin (occurring through alterations in the quinolone resistance-determining region of the genes encoding DNA gyrase or topoisomerase IV) and tetracycline resistance (associated with an efflux mechanism encoded by the tet(B) gene, typically located on conjugative plasmids) remains low (< 2%), resistance to trimethoprim-sulfamethoxazole (resulting from mutations in the dihydrofolate reductase (dfrA) or dihydropteroate synthase (folP) encoding genes, or from the acquisition of sul genes) is more prevalent, reaching 20% in the infection group. Once again, these findings align with data from other European countries.
H. influenzae is a prominent global pathogen responsible for community-acquired respiratory tract infections. Following the implementation of anti-Hib vaccination in the early nineties, a majority of infections now arise from NTHi. The susceptibility of H. influenzae to beta-lactams, specifically aminopenicillins and cephalosporins, is impacted by various resistance mechanisms, and these antibiotics are typically the first choice for treatment. In the current study, several strains exhibited resistance to cefotaxime and/or meropenem, including one invasive strain resistant to both, significantly limiting therapeutic options. Notably, mutations in the ftsI gene associated with high-level resistance (group III) were identified in both carriage and infection strains. The clinical implications of these mechanisms warrant further investigation. National reference centers play a pivotal role in ensuring comprehensive and standardized surveillance of all cases of invasive H. influenzae disease. Regular monitoring of beta-lactam susceptibility is essential to guarantee safe empiric therapy in severe cases and to identify any potential transition from low-level to high-level resistance in the future. Furthermore, WGS analyses should facilitate a more comprehensive understanding of the genetic rearrangements that contribute to the development of AMR in H. influenzae.