Epidemiological analysis of invasive Haemophilus influenzae clinical isolates obtanied from Portugal, Spain and the Netherlands

Background. Haemophilus influenzae is a human-restricted pathogen that forms part of the normal nasopharyngeal microbiota. The introduction of the H. influenzae serotype b vaccine has drastically decreased the number of bacteremia cases caused by H. influenzae serotype b (Hib). Conversely, the cases of non-typeable H. influenzae (NTHi) bacteremia have increased substantially. Therefore, we aimed to perform an epidemiological study comparing invasive H. influenzae clinical isolates from three European countries. Material & Methods. Clinical isolates were obtained from two southern European countries, Spain (Hospital de Bellvitge, n=44; Hospital de Donostia, n=18) and Portugal (n=55), and a northern country, the Netherlands (n=146) between 2013 and 2015. The clinical source of the samples was blood (n=250), cerebrospinal fluid (n=4) and pleural effusion (n=9). Capsular serotyping was done by PCR and genotyping by PFGE (SmaI), followed by FingerPrinting analysis. Antimicrobial susceptibility was tested by disk diffusion and microdilution against Ampicillin (AP), Tetracycline (TC), Chloramphenicol (CL), Ciprofloxacin (CP) and Trimethoprim/Sulfamethoxazole (T/S). Results. Overall, NTHi were the most prevalent isolates (201/263, 76%), followed by Hib (38/263, 14%), Hif (16/263, 6%) and other capsulated H. influenzae (Hia=3; Hid=1; Hie=4). By countries, NTHi was also the major pathogen identified in Spain (82%), Portugal (80%) and the Netherlands (73%), while Hib was slightly more frequent in the Netherlands (27/146, 18%) and Portugal (7/55, 13%) than in Spain (4/62, 6%). PFGE clustering identified high diversity among the NTHi strains, although some strains from different countries were found to be highly related (14 clusters of two or three strains). Hib were grouped together in four main clusters including isolates from different countries: Cluster I (5 strains Netherlands; 4 strains Portugal), Cluster II (18 strains Netherlands, 2 strains Portugal; 1 strain Spain), Cluster III (5 strains Netherlands), Cluster IV (1 strain Portugal; 1 strain Spain).N/