Background: Non-typeable Haemophilus influenzae (NTHi) forms part of the normal nasopharyngeal microbiota in humans, but it is also an opportunistic pathogen causing respiratory infections and bacteraemia. Recently, high molecular weight (HMW1) proteins have been identified as a key factor for cell invasion, a feature implicated in persistence during chronic infection(a). Our aims were to identify the different allelic variants of the HMW adhesin and, given the characteristics of these surface proteins on bacterial adhesion capacity, the second objective was to check if their presence could be related to biofilm formation.
Materials/Methods: A collection of 89 strains isolated from patients with bacteraemia from Spain and Portugal in the 2013-2014 period were used in this study. Strains were genotyped by PFGE (SmaI) and analyzed with the FingerPrinting software (BioRad). The allelic variants of the hmw gene (Hi375 and Hi86-028NP) encoding the high molecular weight adhesins Hmw1/Hmw2 were identified by PCR amplification. Biofilm formation was performed in a static biofilm assay with crystal violet staining. Statistical analysis was performed using the GraphPad Prism 5 software.
Results: Forty-eight NTHi isolates (54%) were positive for the hmw gene. Only the allelic variants of the Hi375 strain could be identified, among them, one strain (1/48, 2%) had hmw-1A, 33 (69%) had hmw-2A and 14 (29%) had both allelic variants, hmw-1A and hmw-2A. Biofilm formation showed great diversity among the studied strains with OD¬570 values ranging between 0.06 and 1.4. Forty-three strains (48.3%) were classified as high biofilm formers and the remaining 46 strains (51.7%) were low biofilm formers. An inverse relationship was found between the presence of hmw genes and in vitro biofilm formation. The invasive NTHi clinical isolates presented high genetic diversity by PFGE, with no strain clustering observed linked to the presence of hmw genes or to biofilm formation.
Conclusion: The allelic variants of the H. influenzae strain 375, especially the hmw-2A gene, were more commonly found among invasive NTHi clinical isolates, which despite having an important role on intracellular invasion, were not linked to in vitro biofilm formation.
(a)Mell, JC et al. (2016). PLoS Pathogens 12: e1005576.N/
