Sub-inhibitory concentrations of ampicillin change the ultrastructure of newly formed NTHi biofilms.

Scanning electron microscopy (SEM) images comparing NTHi biofilms formed in the absence and presence of ampicillin on Thermanox coverslips. A–C: SEM of 2019 biofilms. A) At low power the 2019 biofilm is seen as a mat covering the Thermanox substrate. Bar = 500 µm. B) higher magnification shows the biofilm to be composed of partitions forming empty spaces, or cells, covered with a film of amorphous material (arrow). Bar = 10 µm. C) the partitions within the biofilm are composed primarily of bacterial cells aggregated into flat sheets. Bar = 5 µm. D–F: SEM of 2019 biofilms formed in 90 ng/mL ampicillin. D) The 2019 biofilm formed with ampicillin covers the substrate. Bar = 500 µm. E) The biofilm is composed of partitions around empty spaces with a flat sheet of amorphous material (arrow) over the top. Bar = 50 µm F) The biofilm partitions are composed of aggregated bacteria embedded in sheets of amorphous material. Bar = 5 µm. G–I: SEM of PittGG biofilms. G) The PittGG bacteria form a biofilm over the Thermanox surface. Bar = 500 µm. H) The biofilm is composed of bacterial cells aggregated into poorly defined partitions and covered with a layer of amorphous material (arrow). Bar = 10 µm. I) The biofilm is composed of bacterial cells aggregated into widely spaced strands, and empty space. Bar = 5 µm. J–L: SEM of PittGG biofilms formed in 170 ng/mL ampicillin. J) In the presence of ampicillin the PittGG bacteria form a biofilm comprised of a thick mat, which appears to be strongly attached to itself but less well attached to the substrate. Bar = 500 µm. K) The biofilm mats appear to be composed mostly of amorphous material, a layer of which covers the biofilm (arrow), and arranged into tightly-packed thin partitions. Bar = 50 µm. L) the biofilm is composed of amorphous material formed into thin partitions. The few bacteria detected were embedded in the thin partitions. Bar = 5 µm.