Green fluorescent protein as a novel species-specific marker in enteric dual-species biofilms

Green fluorescent protein (GFP) was used as a tool to examine the interactions between pairs of bacterial species and their effects on subsequent biofilm development over 24 h. A plasmid encoding GFP from Aequorea victoria was transformed into strains of Enterobacter agglomerans and Escherichia coli ATCC 11229. The development of dual-species biofilms, containing one fluorescent and one non-fluorescent partner, was examined using viable counts. UV illumination of plates enabled both species to be identified in a mixture. The spatial distribution of each species was examined by UV microscopy, simultaneously staining the non-fluorescent strain with propidium iodide. GFP fluorescence was measured to quantify the adhesion of the strains to other cells or cell constituents or the invasion into pre-existing biofilms. Co-operation between Ent. agglomerans/GFP and Klebsiella pneumoniae G1 resulted in a 54 and a 23% increase in biofilm formation, respectively, compared with single-species biofilms. E. coli/GFP and Serratia marcescens 87b stably co-existed in biofilms but did not affect the growth of each other. The other bacterial partnerships examined were competitive, with the end result that one species dominated the biofilm. The methods described provide a convenient technique for the examination of mixed-species biofilm communities where the unique interactions between species determine the true properties of the resultant biofilms.