| Summary: | Green fluorescent protein (GFP) was used as a tool to examine the interactions between pairs of bacterial species. A plasmid encoding GFP from <I>Aequorea victoria</I> was transformed into strains of <I>Enterobacter agglomerans (EntGFP) </I>and <I>E. coli </I>ATCC 11229 (<I>E. coliGFP</I>). UV illumination of plates enabled species to be identified in a mixture; their spatial distribution was examined by UV microscopy; and fluorescence measurements were used to quantify adhesion and inhibition of adhesion of the strains to other cells or cell components. Cooperation between <I>EntGFP </I>and <I>Klebsiella pneumoniae G1 (KlebG1) </I>resulted in enhanced biofilm formation and alteration of dual species biofilm properties. <I>E. coliGFP</I> and <I>Serratia marcescens 87b (Serr87b) </I>stably coexisted 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. Microscopic examination of <I>EntGFP</I> and <I>KlebGI</I> dual species biofilms showed that the two species were often closely juxtaposed in microcolonies, suggesting the interactions involved surface associated macromolecules. They appeared to directly interact through adhesin/receptor interactions and proteins were isolated which could form the basis of their specific interactions. In addition, EPS affected coadhesion non-specifically. Compared to single species biofilms, both species in cooperative dual species biofilms had increased resistance to disinfectants and antibiotics. Neutral dual species biofilms of <I>E. coliGFP </I>and <I>Serr87b </I>did not show increased resistance to disinfection. Therefore, the enhanced resistance was related to the specific interactions and disruption of the cooperative partnership though the integration of a third species, <I>Serr87b, </I>led to a decrease in resistance. The methods developed provide a convenient technique for the examination of mixed species biofilm communities where the unique interactions between species determine the true properties and resistance of the resultant biofilms.
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