Abstract

Individual bacteria can accumulate to form biofilm which can help bacteria escape from host immune defense and many antimicrobial agents. Therefore biofilm involves a large number of bacteria which can induce chronic diseases. Here, we compared the differences of biofilm formation among Nontypeable Haemophilus influenzae(NTHi) single culture condition, Pseudomonas aeruginosa (P. aeruginosa) single culture condition and NTHi- P. aeruginosa co-culture condition. Biofilm clone formation unit (CFU) counting revealed that the number of clinical isolated P. aeruginosa was larger than clinical isolated NTHi 3 days after incubation. In co-culture condition, the CFU was significantly higher than both types of single culture condition. Consistent with this result, measurement of crystal violet staining of bacteria attached on the plate well showed that, when co-cultured, the absorbance at 570 nm enhanced 2 - 3 times compared with this two types of single culture condition. Our results were confirmed under scanning electron microscope (SEM). 3 days after inoculation, the co-cultured bacteria stacked more densely to form channels and mushroom-like structure. Whereas both single cultured groups exhibited a more or less sparse, flat-net shape. Intriguingly, both clinical isolated NTHi and P. aeruginosa showed weaker capability in the case of biofilm formation compared with their sibling strains NTHi49247 and P. aeruginosa27853, respectively. Because all the clinical strains were isolated from children whose immuno-system had not yet developed mature or strong enough, bacteria that had less infectious capability were opportunistically invasive. Our results indicate that NTHi and P. aeruginosa can form biofilm in vitro synergistically.

Key words: Nontypeable haemophilus influenzae, Pseudomonas aeruginosa, dynamic observation, biofilm formation