Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea.

Results: During an epidemiologic study focusing on infantile diarrhea, aggregative C.

freundii (EACF) and EAEC strains were concomitantly recovered from a severe case of mucous diarrhea. Thereby, the occurrence of synergic events involving these strains was investigated.

Coinfection of HeLa cells with EACF and EAEC strains showed an 8-fold increase in the overall bacterial adhesion compared with single infections (P <0.001). The synergic effect was mediated by physical interactions among the bacteria and primed in the absence of chemical signaling and without the participation of host cells.

Thus, significant increases (2.7-fold on average) in bacterial adhesion were also observed during the formation of mixed biofilms on abiotic surfaces. Bacterial settling assays showed that EAEC strains harboring F-pili genes (traA) were capable of forming bacterial aggregates only in the presence of EACF.

Scanning electronic microscopy analyses revealed that bacterial aggregates as well as enhanced biofilms formed by EACF and traA-positive EAEC were mediated by non-bundle forming, flexible pili. Moreover, mixed biofilms formed by EACF and traA-positive EAEC strains were significantly reduced using nonlethal concentration of zinc, a specific inhibitor of F pili.

In addition, EAEC strains isolated from diarrheic children frequently produced single biofilms sensitive to zinc.

Conclusions: Putative F pili expressed by EAEC strains boosted mixed biofilm formation when in the presence of aggregative C. freundii.