Biofilm forming P. aeruginosa induces an enhanced inflammatory response in human monocytes

Background

The clinical picture of sepsis is varying, and the severity of the disease is influenced by numerous factors including the infectious agent and the host genetics. P. aeruginosa and S. aureus, the frequently sepsis-causing agents, can switch from a planktonic to a biofilm lifestyle. The cell wall of P. aeruginosa contains lipopolysaccharide (LPS), which can detach and trigger the immune response in the patient. We investigated the effects of live planktonic or biofilm bacteria on human monocytes in terms of the production of proinflammatory cytokines, such as TNF, in order to determine whether these two bacterial lifestyles influenced the immune response during sepsis.

Materials and methods

Clinical isolates of P. aeruginosa and S. aureus, bacterial culture plates and media (Luria-Bertani and tryptic soy broth), RPMI media, ELISA kits, a spectrophotometer and biofermentors were used. The same bacterial concentration of planktonic and biofilm forming P. aeruginosa and S. aureus strains was obtained by optical density measurements and confirmed by colony counting. Blood samples were collected from healthy donors and monocytes were isolated by adherence and further incubated together with live planktonic or biofilm forming strains of P. aeruginosa or S. aureus for 5 hours; the cytokine content was measured by ELISA. LPS was extracted from P. aeruginosa in order to investigate the structural differences between planktonic and biofilm derived LPS. The limulus amebocyte lysate test, SDS-PAGE gel electrophoresis and mass spectrometry of LPS were performed to analyze the LPS structure.

Results

The production of TNF, IL-6 and IL-1α was increased in monocytes incubated with biofilm forming P. aeruginosa as compared with planktonic ones, whereas no difference between cytokine responses was observed in monocytes incubated together with planktonic or biofilm S. aureus. Two predominant forms of rough LPS were detected in planktonic P. aeruginosa by SDS-PAGE and one of the rough LPS bands was absent in the biofilm. Fatty acids differed by their level of hydroxylation in the two bacterial growth conditions as seen by mass spectrometry.

Conclusion

Biofilm forming P. aeruginosa induces an enhanced inflammatory response in human monocytes compared with the planktonic bacteria, and LPS structures were found to be different. No difference was seen in response to S. aureus planktonic or biofilm bacteria. The biofilm P. aeruginosa was more immunostimulatory than the planktonic form. LPS from biofilm forming bacteria may increase the immune response during sepsis.