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Characterization of Haemophilus influenzae non-invasive disease in children, in Portugal: 2015-2022
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Background: Haemophilus influenzae is a Gram-negative bacterium that colonizes the human upper
respiratory tract, where it can remain asymptomatic. It can also progress from colonizer to pathogen and cause acute mucosal infections, such as otitis and conjunctivitis, particularly in children. These infections are frequently associated to NTHi H. influenzae. Empirical treatment with antibiotics is of concern due to possible emergence and dissemina-tion of resistant strains.
Aims / Methods: We aim to characterize H. influenzae isolates from two epidemiologically relevant non-invasive diseases, otitis media and conjunctivitis, in Portugal, from 2015 to 2022 and compare this data with results from invasive disease.
From January 2015 to December 2022, 134 H. influenzae isolates (78-ear-swab; 56-eye-swab) were collected in the National Reference Laboratory for Haemophilus influenzae, based at the NIH, in Lisbon. Most isolates were from children (99.5%; 132/134).
Capsular status was characterized by conventional PCR. Beta-lactamase producers were identified with nitrocefin. Antimicrobial susceptibility was determined by microdilution, according to EUCAST guidelines, for several antibiotics of interest. Genetic diversity was studied by MLST and ST assigned in PubMLST (https://pubmlst.org/organisms/haemophilus-influenzae/).
Results: Among 134 H. influenzae isolates, 99.3% were NTHi (133/134), whereas only one encapsulated isolate was found, and characterized as Hia (0.8%, 1/134). Beta- lactamase producers accounted for 6.7% (9/134). Antibiotic susceptibility results (n=113) showed that most isolates were susceptible to the antibiotics tested, with the exception of 34.5% resistance to trimethoprim-sulfamethoxazole (39/113). In the course of this study, we highlight the characterization of a beta-lactamase negative, NTHi isolate, resistant to ampicillin, cefotaxime, cefuroxime, amoxicillin-clavulanic acid, cefepime, and trimethoprim/sulfamethoxazole. This is the first time that we characterized resistance to cefotaxime in H. influenzae, in our country. The isolate, from a 67 years old man with multi-microorganisms corneal ulcer, was characterized as BLNAR group III-like, ST3 (confirmed by WGS).
High genetic diversity was observed among NTHi, as expected, with 22 different STs assigned for 31 isolates (71% 22/31), although ST12 and ST34 included three isolates each. When comparing the MLSTs results of isolates from both invasive and non-invasive diseases, we observed that 41% (9/22) of the STs were shared among both diseases: ST-12, ST-34, ST-142, ST-160, ST-262, ST-367, ST-396, ST-1034, and ST-1411.
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Haemophilus influenzae Serotypes Hib Vaccine Otitis Conjuntivitis MLST Antimicrobial Resistance Children Resistência aos Antimicrobianos Infecções Respiratórias Portugal