Summary auto-generated
This study used green fluorescent protein (GFP) as a species-specific marker to examine interactions between pairs of enteric bacteria during dual-species biofilm formation. Researchers transformed GFP plasmids into Enterobacter agglomerans and Escherichia coli ATCC 11229, then studied their interactions with Klebsiella pneumoniae and Serratia marcescens over 24 hours. Using viable counts, fluorescence microscopy, and fluorimetry, they identified three interaction types: cooperation (Ent. agglomerans/GFP and K. pneumoniae showed 54% and 23% increases in biofilm formation, respectively), coexistence (E. coli/GFP and S. marcescens formed equal biofilms regardless of partner presence), and competition (E. coli/GFP dominated K. pneumoniae; S. marcescens dominated Ent. agglomerans/GFP). Microscopy revealed cooperative species formed closely associated microcolonies, while competitive species formed discrete ones. Adhesion studies suggested cooperation involved both protein and polysaccharide-mediated interactions. GFP proved stable through cell lysis and remained fluorescent after cell death, enabling spatial localization but limiting use as a viability indicator. These findings demonstrate GFP's utility for tracking species-specific behavior in complex biofilm communities.
Key findings
- Ent. agglomerans/GFP and K. pneumoniae cooperated, increasing biofilm formation by 54% and 23% respectively compared to single-species biofilms
- E. coli/GFP and S. marcescens coexisted stably without affecting each other's growth
- Competitive pairs (E. coli/GFP with K. pneumoniae; S. marcescens with Ent. agglomerans/GFP) resulted in dominance by the faster-growing species
- Cooperative adhesion between Ent. agglomerans/GFP and K. pneumoniae involved both protein adhesins and polysaccharide receptors
- GFP remained fluorescent after cell lysis and death, useful for localization but not suitable as a viability marker
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Abstract
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.