Increased Biofilm Formation by Nontypeable Haemophilus influenzae Isolates from Patients with Invasive Disease or Otitis Media versus Strains Recovered from Cases of Respiratory Infections

Abstract

f Biofilm formation by nontypeable (NT) Haemophilus influenzae remains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NT H. influenzae by comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NT H. influenzae isolation, with significantly increased biofilm formation for NT H. influenzae isolates collected from patients with invasive disease and OM compared with NT H. influenzae isolates from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.

Effects of Low-Dose Amoxicillin on Staphylococcus aureus USA300 Biofilms

“…After 3 h, the wells were magnetized using a block of 96 magnets in standard microplate format. Under these conditions, microbeads migrate to the center of the well, forming a spot, but bacterial cells attached to the bottom of the well block the migration of the beads and reduce the intensity of the spot (21). Figure 3A shows photographs of wells that were inoculated with sterile broth, JE2 inoculum, or JE2 inoculum supplemented with low-dose amoxicillin and then incubated for 3 h and magnetized.…”

“…Under these conditions, free beads migrate to the center of the well, forming a spot. Bacterial cells that attach to the bottom of the well block the migration of the beads and reduce the intensity of the spot (21). The plate was imaged using the BioFilm Control plate reader and analyzed with the accompanying image analysis software.…”

Critical review on biofilm methods

“…While this may be appropriate for (large scale) screening purposes, other investigations may require specific hydrodynamic conditions, substratum composition or large quantity of biomass material that the microtiter tray based methods cannot provide. The choice of the device depends on the scientific question and a complete characterization of biofilm formation ability for a strain generally requires combining different approaches (Puig et al, 2014;Renier et al, 2014).…”

Bacterial Lysis through Interference with Peptidoglycan Synthesis Increases Biofilm Formation by Nontypeable Haemophilus influenzae

“…The speed of initial bacterial adhesion was evaluated by the biofilm ring test on modified 96-well polystyrene plates obtained from BioFilm Control (St. Beauzire, France) as previously described (13). Briefly, bacterial suspensions were mixed with magnetic beads, incubated for 2 or 4 h at 37°C, and placed on a magnetic block.…”

“…For instance, we found that especially NTHi strains collected from patients with OM have the ability to form biofilms in vitro (13). This is consistent with in vivo experiments, where biofilms in the ears of chinchillas were associated with bacterial persistence (14).…”