Background: We have recently demonstrated that P. aeruginosa planktonic cells survive β-lactam antibiotic exposure (meropenem) by transitioning to L-forms but they can efficiently be killed by antimicrobial peptides (nisin). The aim of this study was to investigate whether the morphological transition occurs also in biofilms and the combination with an antimicrobial peptide could be effective in eradicating P. aeruginosa biofilms.
Methods: Mature biofilms were grown for 3 days in M9 minimal medium using the flow cell technology, treated with meropenem for 8 h, 16 h and 24 h and stained with a membrane dye for morphology analysis. Moreover, flow biofilms were treated with a combination of meropenem and nisin for 24 h, stained with a membrane/dead cell dye mixture for quantification of biofilm biomass. The morphological conversion and the combination treatment were also investigated in biofilms grown in artificial sputum medium. Samples were visualised with deconvolution microscopy, images were processed using Imaris and biofilm biomass quantified using COMSTAT.
Results: L-forms could be detected in meropenem-treated samples after 16 h and 24 h of treatment. The combination treatment determined a significant decrease in total biofilm biomass and a significant increase in dead biomass after 24 h of treatment compared to untreated and single treatment samples. Moreover, the dead biomass remained trapped within the biofilm matrix after the combination treatment.
Conclusion: We showed for the first time that bacteria in biofilms convert to L-forms when exposed to a β-lactam antibiotic and that a combination with antimicrobial agents could provide a novel strategy for treating P. aeruginosa infections.